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Biacore T100 - GEヘルスケアバイオサイエンス

Contents

1. 4 1 lt 0 5 TFA 0 1 ZipTip Milipore 50 mM NaOH RE NaCl Surfactant P20 DMSO
2. O08 parE Quality Control Report Residuals Parameters eo Kinetic constants are within instrument specifications o Kinetic constants appear In be uniquely determined o No significant bulk contributions RT Faund o Check that sensorgrams have sufficient curvature OO amp o oO o Examine the residual plot Pay attention to systematic and non random deviations ka 103 107 1 Ms kg 105 0 5 1 s ka ka Rmox k k ORURO R
3. Biacore T100 234 5 5 8 1 Toolbar Run Method S Menu bar Run Method Te Open New Method Look in E Methods nd Templates Name Modified Biacore Methods Biacore Methods Te Open New Method Lookin Biacore Methods Name Type Modified Eb Affinity in solution Method Builder 3 28 2008 5 Calibration Free Concentration Method Builder 3 28 2008 5 GST Kinetics Method Builder 3 28 2008 5 Inject and recover Method Builder 3 28 2008 5 Kinetics heterogeneous analyte Method Builder 3 28 2008 5 L1 liposome capture Method Builder 3 28 2008 5 LMW kinetics Method Builder 3 28 2008 5 LMW screen Method Builder 3 28 2008 5 NT kinetics Method Builder 3 28 2008 5 Single cycle kinetics Method Builder 3 28 2008 Inject and recover Open Method Builder D Main
4. Assay Steps Cycle types Assay Steps Startup Samples Control Samples Cycle types Assay Steps Cycle Types Biacore T100 248 6 6 1 _ Toolbar Run Method Menu bar Run gt
5. Eject Rack Tray Hack Tray Ejected Click OK to retur the rack tray In the sample compartment OK Time to auta close 50 Eject Rack Tray 60 OK Biacore T100 12 1 1 10 Ja NN O TT T DTEROSIT Reagent rack Type 1 15ml 11 mm x20 Reagent rack Type 2 16 mm 4m x9 15mm 4 ml x9 7 mm
6. Biacore T100 4 51 New Cycle 1 l End Manual run 4 Menu bar Commands gt End Run Standby flow E 4 3 Tool bar Reference Line 3z debe View Reference Line
7. x Life Sciences Academy 71 3415 35 ISO 9001 2008 TEL 03 5331 9336 FAX 03 5331 9370 e mail Tech JPG ge com
8. RU RU 40x1 Sx RU 200 x 1 Sx S 50 kDa Pyo24bkon cs 100 kDa 1 40 x 1 1 x 50 0007100 000 20RU 200 x 1 1 x 50 000 100 000 100 RU 20 100RU Biacore T100 3 25 3 1 N g CM NHS N NHS
9. 5 0 2 _ 1 2 n 2 10 0 30 ul min 2424 FUE CREE 2 2 5 10 t EILSZeCRUM se ARRENDE RAOS 731a 10 30 Biacore T100
10. Solvent correction Extrapolate solvent Correction Extrapolation Range Extrapolation Range asis 1000 RLI 10 OK RU 10 Zoom loc 5 ME CY 0 yoy NS 5 T E 4 L3 F A amp 40 y i a 15 20 25 2000 1500 1000 500 0 500 1000 1500 2000 2500 Response Ref Biacore T100 230 5 5 5 5 1
11. Ctrl Breakdl Biacore T100 Evaluation Software Biacore T100 5 183 5 5 2 Evaluation 74x Sensorgram Fusco ER di P All sensorgrams PS R dit J Remove_ Edt Curve Name Fc 4 3 JP e Sensorgram aJ All sensorgrams RU gt Plot 8800 C Zoom Lock Baseline Sample Binding level Binding stability Binding to reference Controls binding 8400 Controls stability E Report Point Table 8200 mj Report Point Table Conditioning Control Sample Sample Startup 7600 7400 7200 7000 6800 0 Time 5 56
12. 4C 50 ml HBS EP 4 o Sensor Chip 4 mum S BIACORE Sensor Chip Qr is n 6 CM5 ido Biacore T100 8 REDAS S 291 Fs UL TEY F9 50 IBDREUOD ODE
13. Kp M 1 Ko Ka 1 M A BAB Kp A B AB Biacore Koz ka kc ko A Mis AFRE k s1 Ko A B AB Ko ka ka KA Ka kq Biacore T100 5 55 Kp ka ko
14. Dock Insert Chip Cancel Toolbar Eject Ex Eject Chip Insert Chip Toolbar Insert Biacore T100 1 7 HE 1 6 Reuse Chip Insert Chip Mew chin 9 Reuse chin Reuze chip Reuse CMS Id 080507 1029 12114 Chip type CM5 Chip id pansz 1023 12114 Chip lot na Reuse id Details Chip
15. CFCA QR f M k ConO arya 20min dt T N T Pd Pod P 64 d 5ul min Mw 4s Da Kn CFCA 4338 150kDa 5000RU Mw km Con d R dt 2 5 100ul min km D
16. RU 5 0 Biacore T100 264 6 6 3 Solvent correction Cycle types New New D General settings Cycle types Thermodynamics aiz Mew Conditioning dcc g amp Delete CT Sm i 5 Copy Cycle types Thermodynamics Cond
17. S Recurrence Calibration Control Sample Solvent correction 6 1 Assay step preparations img DAZ 27 7298 uRODEHRed 425 25727 72742k x9 1 A Number of replicates As Entered Order Random Biacore T100 6
18. T 060327 Thermo Rack Positions Reagent Rack 2 Type Sample 1 1 148 Negative control Control sample 40 i 148 Negative control Control sample 148 Negative control Control sample i 148 Negative control Control sample i 148 Negative control Control sample E j OGO 148 Negative control Control sample 1 Oe 148 Positive control Control sample A F G i 148 Positive control Control sample 96 Deep Well Microplate 148 Positive control Control sample 148 Positive control Control sample 148 Positive control Control sample E e Q L Q O O Q O 148 Positive control Control sample T e Q O O C 148 Analyte A Sample 10 i 148 Analyte A Sample n OQ e C C C C C C i 148 Analyte A Sample o Q s Q Q s 148 Analyte A Sample 9 Q Q Q Q O O Q OQ 148 Analyte A Sample 7 O Q Q O O O O O 148 Analyte A Sample i 148 Analyte A Sample e e Q Q Q Q C 148 Analyte A Sample o Q Q O Q 148 Analyte A Sample Y Q Q amp Q O C O 148 Analyte A Sample i 148 Analyte A Sample 148 Analyte A Sample 148 Analyte A Sample 148 Analyte A Sample eMe Back Next gt Menu 7j 5 Automatic Positioning amp 338 5v Te Automatic Positioning Change the order in which the samples are positi
19. RU Adjusted sensorgram ability eabilty g ly S Response 0 baseline a E 5 n E i FA 7 g 100 50 0 50 100 150 200 250 300 350 Time 0 baseline Sensorgram window _ cok Tools gt Sensorgram Adjustment Biacore T100 302 9 Y Adjustment les i O Injection Event response 0 Enable Second Adjustment Mormalize O Report Point response 100 Injection Event response 100 Ens Ea Enable Second Y Adjustment Normalize Injection Event respons 100 3 Sample 1 stop OK RU Adjusted sensorgram 120 Response 0 baseline 100 Sarrple 1 stop 100 50 50 100 150 200 250 300 350 Time 0 baseline s 100RU Biaco
20. co binding stability co binding co stability e Sample hi Carry over control Regeneration HE 5 76 Wizard Binding analysis Kinetics Affinity Wizqard 3
21. Biacore T100 120 5 5 28 FS Kinetics Affinity Fit Kinetics Create Curve Fc 2 1 Ligand antibody 1 ug ml pH5 Sample antigeni Temperature 25 C Add Fit Current Fits Model 1 1 Binding v E j Description ka 1 Ms kd 1 s KD M Rmax RU Conc M tc 1 531E 6 0 002610 1 705E 9 7 178E 7 1 100E 9 2 231E4 8 2 200E 9 2 231bE 8 X 0 2011 4 300E 9 2 231E4 8 0 1965 8 500E 9 2 231E4 8 0 1167 1 700E 8 2 231E 8 0 1062 Finish Add Fit Model Fit LA Kinetics Affinity Fit Kinetics Create Curve Fc 2 1 Add Fit Model Two State Reaction css Report Residuals Parameters kal 1 Ms kd1 1 s ka2 1 s kd2 1 s KD M Rmax RU Conc M tc Flow ul min kt RU Ms 1 554E 6 0 002932 5 060E 4 1 86
22. OANA HE SP e c6REOXS e5X33J2 QU e o lE Pe OFF
23. s Enhancement 2 Regeneration 40 High viscosity solution Biacore T100 6 261 Carry over control 40 ul min 30 Evaluation Software Solvent correction 30 ul min 30 5 7
24. 2 Next gt Biacore T100 5 103 Te Method Builder Cycle run list buffer buffer buffer antigen antigen antigen gt Next gt Te Method Builder System Preparations Frime before run Normalize detector zs zs Prime Normalize Temperature settings Analysis temperature 25 C Sample compartment temperature 25 C Next gt Biacore T100 104 5 T Method Builder Rack Positions Sample 1 CP contene Tree swte 1 cone Gr ea 118 antigen Sample O 11500 118 antigen Sample 118 antigen Sample 118 antigen Sample 118 antigen Sample 118 antigen Sample 118 antigen Sample 118 antigen Sample 118 antigen Sample 118 antigen Sample 118 antigen Sample 118 antigen Sample 118 antigen Sample 118 antigen Sample 118 antigen Sample 154 buffer Startup
25. Rm ARAS 20RU Rmo x xS Da RU Da S 50 000 Da 1 000 RU 1 20 000 Da Ras 20 000 x 1 000 50 000 x 1 400 RU 10 000RU RU vs C ka ka Ko
26. Concentration Analysis Create Control Samples Concentration Analysis Create Calibration Control Samples Samples Control 5 ample T able Control 5 ample Plot najml Conc Response Calc Conc 40 Cycle Sample Id ng ml RU ng ml 10 Control 1 33 3 i 33 27 17 Control 1 33 3 33 15 24 Control 1 33 3 i 34 78 31 Control 1 33 3 32 69 44 Control 1 33 3 34 01 51 Control 1 Avg f 100 6 co e 11 Control 2 i i 100 1 18 Control 2 f i 101 2 25 Control 2 f i 103 8 32 Control 2 1 102 3 45 Control 2 f i 102 3 52 Control 2 E g B B O B 3 3 102 1 5 39 Tools Custom Report Points 29 Y v 7d 5S Custom Report Points Custom Report Paints Add Biacore T100 142 5 ddd Custom Report Point Report point ld stability2 yindo s Fosition the report point seconds Sample 1 injection stop vt Calculate response relative to report point Cycles Apply To Selected Assay step purpose Selected Assay step purpose Startup C Appl To S
27. crude Bulk Effect Se HLTA f amp ewl 07724 REOR CRRERESRI 7j 3 e SEG RABAT FOREZ BSHT KGSRIC RE SAxSu3BEMH 6S ng ml ug ml
28. IESU fO Incubation time Incubation time ONS ROAS s Deposition solution Deposition solution volume Ul Number of repetitions 1 10 4 5 Flow rate i Biacore T100 240 5 Conditioning T Method Builder Main Cycle types ect and Recover General Settings g Conditioning Assay Steps Cycle Types i 2 Variable Settings Commands Report Points Capture v mm MILL ej t Description of selected cycle type Conditioning of the surface is performed by in
29. Y 100 Y 0 CX OK Biacore T100 5 189 Evaluation Add Report Point Table 2 v 2d 2 Evaluation Explorer Report Point Table 2 Work qreq P AU w Biacore T100 Evaluation Software C Documents and Settingsibiacore DesktopiT100Manuali Ch_drug scr bme Report Point Table z File View Evaluation Tools Window Help x i a ded 5ensoraram Plot mi Bar Chart A3 Kinetics Affinity Concentration Analysis Thermodynamics Evaluation Explorer n x Remove C Window s F gt Sensorgram binding All sensorgrams binding 8363 5 D 02497 E Plot binding 8321 6 0 08206 Baseline Sample binding 8281 4 0 1026 a 1 Binding level binding 8242 4 0 04047 Binding stability a Binding to reference a Controls binding
30. Menu Simple Position Import Biacore T100 220 5 5 73 Control Sample R1A1 R1412 12 060327 The Reagent Rack 2 9 rmo Rack Positions i OeO0O0O0000 148 Negative control 148 Negative control 148 Negative control 148 Negative control 148 Negative control 148 Megative control 148 Positive control 148 Positive control 148 Positive control 148 Positive control 148 Positive control 148 Positive control Control sample Control sample Control sample Control sample Control sample Control sample Control sample Control sample Control sample Control sample Con
31. Overview 6 2 General Settings 72 Verification 5 Over view General Settings Biacore T100 250 6 Assay Steps Cycle Types Variable Settings FRA DIEDE Verifivcation Over view T Method Builder Main Concentration unit nM General Settings Startup Data collection rate 10Hz Sample compartment temperature 25 C Startup LMW kinetics 3 times as entered Detection Dual Assay Steps A Sample Cycle Types Sample LMW kinetics 1 time as entered Settings for assay step Startup Temperature 25 C Variable Settings Solvent correction Buffer t Solvent correction Solvent correction 1 time a
32. RU Adjusted sensorgram 30 25 binding tty stabilty ability ability foi Response 0 baseline S tbinding amp binding A amp limalimg binding x stability baseline e amp binding stability 50 50 100 150 200 250 300 Time 0 baseline Sensorgram window _ cck Tools gt Sensorgram Adjustment Blank Subtraction 9 Enable Blank 5ubtraction Blank Subtraction Enable Blank Subtraction Biacore T100 9 301 RU Adjusted sensorgram g ly Stability 4abilty Response 0 baseline a amp stability amp stabilitystability zs p 8 100 50 50 100 150 200 250 300 350 Time 0 baseline 9 3 6 100
33. R f ks kg Rmax C k kg Req CDD Req vs C C Req 0Z70vHF25 BRER KA Ko Req C X Rmax C Ko Req RU E C M M Biacore T100 56 5 Pwt FiRIE Kp 1 10710
34. Save as Methods and Templates 7 Bia Users Don t Save Save in File nme Save Standby flow l Biacore T100 Evaluation Software Biacore T100 5 _ 133 5 33 Run Stop Run Biacore T100 AN This will stop the run stopRun Kun Stopped Finishing current cycle please wait Abort cycle hn Ei Break _
35. LED 277 ready L temperature 1 1 Sample compartment door f Sensor chip port Sample compartment tm injection window Rack tray port 2UL UZTSRUR tuia Biacore T100 2 1 1 1 2 _A 423 7 BERUF A B C D 4 IU
36. ERD 5 S E RU c Z2 SEI ODfGG 5 d OBERIDREE OEUVRE AUS ZENT S5 87 DAH AS UDEPFERMGSOZ 1 2 1 SRM 5 fifteen no T 29 C Qo 9 37 C Q 45 C lt b cc J Ko kg ka tmi lc dU CHE UTE EREXESA Kp kz k Biacore T100 194 5 vant Hoff Ko
37. Teo v Adjustment for controls Calor By k Sort k IF Fitting k Adjustment for controls Ranking Table Columns Biacore T100 188 5 M Adjustment for controls Plot definition Report point binding Response type Relative response Variable Cycle number Adjustment settings Use adjustment For controla Positive control CB 5A w Negative contral Fitting function Linear O Folnomial Before adjustment After adjustment en e e e i E 5 g I z S E T m E E T m 20 30 40 20 30 40 Cycle number Cycle number Adjustment settings Use adjustment for controls Positive control Negative control Y 0 RU Fitting function Linear Polynomial
38. Biacore T100 132 5 Eject Rack Rack tray port OK Eject Rack Tray Rack Positions Next gt Te Concentration Analysis Prepare Run Protocol Prepare Run Protocol Make sure the correct sensor chip is docked Make sure all samples amp reagents are loaded in the rack and microplate according tn the Rack Positions setup vials should be sealed with rubber caps and microplate with adhesive foil Place the buffers on the left hand tray and insert the correct tubes Mote Standby after run will use buffer A Make sure there is fresh water in the water bottle on the right hand tray If necessary empty the waste bottle before start of the run Estimated run time 9h 54 min excluding conditional statements temperature changes and standby How Estimated buffer consumption Running buffer Appraszimately 300 ml Start
39. AG RTInKp AG AH TAS InKp AH RT AS R I AS R intercept on Y axis AG kJ mol In Kp R T K AH R slope Kp M AH kJ mol AS J K mol I T RTInKp AH TAST AC T To TACpIn T To AC kJ K mol To 25 C Eyring ko ka k keT h exp AS R AH RT Ink T AS R AH RT InkB h k e e IRE AER Me AS R In kg 7 intercept on Y axis kg DII Ev zt h J27253 AS IYORE Ze Q K mol R TAEZ AH mo W d T K AH R slope AG kJ mol I T Biacore T100 5 195 5 6 1 Toolbar Run Wizard Menu bar Run gt Wizard 2s Te Run Wizard Template Lookin Methods
40. AIBCD A doge BCD isl IG U CtSXE OD 7 7 7tEG Re Uv C amp S 500 ml 2 Biacore T100 1 3 1 3 2UI UZtTERGBRICU C WEtE7 5 HBS PBS HBS EP 10X 1000 ml BR 1006 69 0 1 M HEPES 1 5 M NaCI 30 mM EDTA 0 5 96 v v Surfactant P 20 10 fe 83h 0 01 M HEPES 0 15 M NaCl 3 mM EDTA 0 05 96 Surfactant P 20 pH7 4 HBS P 10X 1000 ml BR 1006 71 0 1 M HEPES 1 5 M NaCI 0 5 96 v v Surfactant P 20 10 0 01 M HEPES 0 15 M NaCI 0 05 96 Surfactant P 20 pH7 4 HBS N 10X 1000 ml BR 1006 70 0 1 M HEPES 1 5 M NaCI gt T 10 0 01 M HEPES 0 15 M NaCl pH7 4 PBS 10X 1000 ml BR 1006 72 0 1 M phosphate Buffer 27 mM KCI 1 37 M NaCl 10 5 v v DMSO 0 01 M phosphate Buffer 2 7 mM KCI 0 1
41. Move Up Move Down Startup 1 Startup Thermodynamics 5 times as entered Thermo 1 Sample Thermodynamics 1 time as entered Solvent correction 1 t Conditioning Solent correction 1 time as entered Ewery 15 cycles Control sample 1 t Control sample Thermodynamics 1 time as entered Every 15 cycles 30 Startup 1 Solvent correction 1 Control Sample 1 Thermo 1 1 15 Solvent correction 1 Control Sample 1 Thermo 1 16 30 Biacore T100 6 257 6 2 Recurrence Number of replicates
42. Pooling _ Yes OK Automatic Positioning Biacore T100 64 5 Eject Rack Rack tray port OK Eject Rack Tray Rack Positions Next gt Te Kineticsi ffinity Prepare Run Protocol Prepare Run Protocol Make sure the correct sensor chip is docked Make sure all samples amp reagents are loaded in the rack and microplate according to the Rack Positions setup viales should be sealed with rubber caps and microplate with adhesive foil Flace the buffer s on tha left hand tray and insert the correct tubing s see below Mote Standby after run will use buffer A Make sure there is fresh water in the water bottle an the right hand tray If necessary empty the waste bottle before start of the run Estimated run
43. Menu Simple Position Import 5 54 Control Sample R1A1 R1412 12 Te 060327 Thermo Rack Positions Type Sample 1 148 Negative control Control sample 40 i 148 Negative control Control sample i 148 Negative control Control sample e e i 148 Negative control Control sample QO OO i 148 Negative control Control sample E CX 148 Negative control Control sample i OG GO 148 Positive control Control sample A B F E i 148 Positive control Control sample 96 Deep Well Microplate 148 Positive control Control sample 148 Positive control Control sample 148 Positive control Control sample E o Q O Q O e Q O 148 Positive control Control sample T E Q O O O O O O 148 Analyte A Sample 10 i 148 A
44. 5 10 4ElS 2e xl RBEXREIJIJE C BEREDRISIODIBE 73 Ia c v C Eas Ce 73 MG 10 30 Biacore T100 94 5 5 2 1 Toolbar Run Method S Menu bar Run Method 2s Te Open New Method in EEEE Templates Modified Biacore Methods Biacore Methods Open Te Open New Method Lookin 9 Biac
45. T 060327 Thermo Rack Positions Reagent Rack 2 1 148 Negative control Control sample 40 i 148 Negative control Control sample i 148 Negative control Control sample e e i 148 Negative control Control sample OQO OO i 148 Negative control Control sample E j OGO 148 Negative control Control sample OG GO 148 Positive control Control sample F 6 i 148 Positive control Control sample 96 Deep Well Microplate 148 Positive control Control sample 148 Positive control Control sample 148 Positive control Control sample E e Q L Q O O Q O 148 Positive control Control sample T e Q O O C 148 Analyte A Sample 10 i 148 Analyte A Sample n OQ e C C C C C C i 148 Analyte A Sample o Q s Q Q s 148 Analyte A Sample 9 Q Q Q Q O O Q OQ 148 Analyte A Sample 7 O Q Q O O O O O 148 Analyte A Sample i 148 Analyte A Sample e e Q Q Q Q C 148 Analyte A Sample o Q Q O Q 148 Analyte A Sample Y Q Q Q O C O 148 Analyte A Sample i 148 Analyte A Sample 148 Analyte A Sample 148 Analyte A Sample 148 Analyte A Sample DELE eMe Back Next gt Menu 73 5 Automatic Positioning 355v Te Automatic Positioning Change the order in which the samples are positioned by ordering the regions The first region in the list is positioned first Region Color Orientati
46. Tools snlwent_3 iaraup By e Color By k Shaw Column Labels Biacore T100 5 193 5 6 Biacore AG H AS AG A AH amp AS 2
47. Move Up MoveUp Move Down Move Down Delete XK Delete 2 Base settings Name Purpose Biacore T100 254 6 Purpose Evaluationsoftware 7 f amp Conditioning Startup Solvent correction Calibration Sample Control Sample Undefined Connect to cycle type Cycle types
48. 2 1 4 5 Chip Chip type Capture 2 4 Sample Regeneration 1 2 Carry Over Next gt Thermodynamics Setup Conditioning Run conditioning cycle Solution Contact time s Number of injections Startup Bun startup cycles Solution buffer Number of cycles 3 v Solvent correction Bun solvent correction Number of injections Repeat after sample cycles Temperatures Analysis temperatures Sample compartment temperature Vary with analysis Temperature 4 temperature Biacore T100 5 197 Conditioning Sol
49. Single cycle kinetics 93 Calibration Free Concentration 143 LMW screen 247 Inject qnd recover 233 Biacore T100 54 5 5 1 1 1 VERO
50. Control Sample R1A1 735 R1A12 12 DOOOOOOOOOOO b Menu Automatic Positioning 335 5v T Automatic Positioning Change the order in which the samples are positioned by ordering the regions The first region in the list is positioned first Region Color Orientation Anchor Rack ial Si i First Sort By Bottom left Sample Small Ascending Bottom left Sample Small i Ascending Bottom left Sample Small Ascending Bottom left Reagent Large Content Ascending Bottom left Reagent Small J Content Ascending 4 Control sample E Cyan Column Sample E CarkElue Column Startup I Crimson Column Wash Yelaw Column 4 4 14 4 Solvent correction buffer amp Il Blue Column lt gt ol Auto
51. Biacore T100 Evaluation Software C Documents and Settings biacoreDesktopt BcT100v21CFCATProtA lgG bme m BE 5 Curve Name Fc 4 3 e Assay Step Purpose verlay e Cycle verlay JP ensorgram j All sensorgrams RU Sensorgram gt Plot Baseline Sample Binding level Binding stability Binding to reference Report Point Table mj Report Point Table gt Concentration Analysis LA Protein A mouse IgG C Zoom Lock Time 5 45 Keyword table Tools Keyword Table S Keyword Table Reset All Filters tartup Add Keyword Marfarin i Rename Keyword Warfarin Warfarin Remove Keyword olvent correction Warfarin Warfarin Warlarin ring Warfarin IWarfarin 3828 7 io Warfarin 138 PN gt i Naproxen Naproxel Naproxen Naproxen Naproxen Naproxen lt Naproxen Furosemide Furosemide Biacore T100 166 5 5 46 Menubar Toolbar E Report Point Table
52. l REI Eld Standby flow Biacore T100 Evaluation Software DB 5 65 7072 LORA Run gt Stop Run amp 2 v 2d 2 Hiarnrg T100 AN This will stop the run stop Run Kun Stopped Finishing current cycle please wait Abort cycle hn Ct Break Ctrl Breakl 4 Biacore T100 Evaluation Software Biacore T100 5 203 5 6 2 Evaluation Biacore T100 Evaluation Software Thermodynamics antibody ys antigen blr Concentration Analysis v Curve Nam
53. Thermo 1 Startup Thermodynamics 5 times as entered J Thermo 1 Sample Thermodynamics 1 time as entered Control sample 1 Control sample Thermodynamics 1 time as entered Ewery 15 cycles t 1 Thermo 1 Every lt Distribute Run assay step once first Biacore T100 256 6 Run assay step once first Recurrence Repeat assay step within 9 Every 15 cucle Distribute Ej accurences evenly Run assay step once first Run assay step ance last Cycle Run List 6 2
54. 5 48 Ba Next gt Biacore T100 5 171 5 48 KS Calibration free concentration Select Data Create Settings CO All sample series Single sample series Sample E553 1000 mes Curve Fe 2 1 J E553 1 000times Fc 2 1 Fi Zoom lock lt Back Next gt Cancel Remove Selection Undo Calibration free concentration Evaluation Result Create m Ligand Sample Dilution Meas Conc S QC factor M ratio Fc 4 3 100ug m
55. Cycle Run List ETT Genesi Setigs Cydelwes Heledhunes Variable Settings veis Recurrence Number of replicates Cycle Run List Cycle Run List Simulation Tool Enter the number of cycles you plan to run for each assay step in the left panel The list of cycles for the run is displayed in the right panel Assay Step Name Cycles Assay Step Startup 1 Sample Solvent correction Control sample Cycle Run List Simulation Tool Cycle Assay step name Assay step purpose Cycle type Ol nn rwWINT Startup Startup Startup Solvent correction Control sample Control sample Sample Sample Sample Sample Sample Sample Sample Solvent correction Control sample Control sample Startup Startup Startup Solvent correction Control sample Control sample Sample Sample Sample Sample Sample Sample Sample Solvent correction Control sample Control sample LMW kinetics LMW kinetics LMW kinetics Solvent correction LMW kinetics LMW kinetics LMW kinetics LMW kinetics LMW kinetics LMW kin
56. Include Cycle Biacore T100 140 5 HE 5 37 RU Biotin 1600 1400 A 1200 Exclude Curve 1000 Exclude Cycle Shaw Sensorgram 500 k Labels e 600 Scale Copy Graph 400 Export Cr ves i aridlines 200 Legend E 20 zu 4n B an 100 120 Concentration numi Exclude Curve l Calibration Table Conc Curve Overlay R RU Biotin 1600 1400 2 1200 1000 800 Relative Response 600 400 Parameters 200 lt Dverlay gt 20 0 20 40 En 80 100 120 Concentration najml Include Curve Biacore T100 5 141 5 38
57. Exclude point Exclude curve Shaw Sensorgrams Scale Copy Graph Export Curves Exclude point 7 v 2d 2 15 600 600 400 200 O0 200 400 600 00 1000 1200 Response Ref RL Biacore T100 228 5 5 80 Solvent correction Include 4 Included Cycle Curve Chi RU YD RU 500 600 400 200 0 200 400 600 800 1000 1200 Biacore T100 5 229 5 81 DMSO DMSO DMSO
58. 6 3 Report Points Commands Commands Report Points Capture Sample Enhancement Regeneration Sample 1 L ar over control Carry over control 1 Solvent corector Inject amp ndHecaver General IF then Insert A inset Biacore T100 6 259 54o 4 9 Capture Sample Regeneration Sample amp Evaluation software fere xtd eisciR Jv 7 E COS Sample Types Sample solution contact time Dissociation time Flow rate Flow path
59. A A X A A B B A Calibration Curve Conc ug ml RU 600 570 540 510 g 480 amp 450 7 420 B 390 IL 660 630 10 20 30 40 50 60 70 80 g0 100 110 Concentration units ml Biacore T100 124 5 5 3 1 Concentration Analysis
60. Toolbar Add Report point BE Menu bar Edit Report point Add Report Point Report Paint amp Time 255 0 s Ms Baseline Add to all curves in this cycle Cancel Id 0 Baseline OK Biacore T100 22 2 2 1 3 End Manual run E Menu bar Commands gt End Run Standby flow 2 2 Menubar File gt Save 2 3 File Print OK Print Frnter Printer Micrasaft PS Document riker File Properties File Properties Wizard Te
61. 30 1 E 4 1 1 AES pH a CLCLCCOL LO LE LLLLIITYYTT NaCl lt 2 M 10 mM Gly HCI HCI Phosphoric acid Formic acid PILDI iH 10 mM Gly NaOH NaOH Ethanolamine Ethanolamine HCI EDTA FED ERI Surfactant P 20 Tween 20 Triton X 100 SDS Octylglucoside Acetonitrile 2096 DMSO 896 Ethylene glycol in HBS Buffer 5096 Ethanol 2096 Formamide 4096 zz VERI Guanidine HCI Urea Biacore T100 4 NV 37 AREER FRORA 4
62. Custom Methods Amine Te Custom Methods Methods Aldehude Tes Amine Biacore T100 36 3 Copy Methads Copy of Amine Methods Method name Command Solution Contact Time s Flow Rate ul min ps ps PRE COPC Specified in Immobilization Setup nia PIIXINJECT EDC NHS 50 50 420 10 P WASH Ethanalamine LIGAMDIMJECT Specified in Immobilization Setup 4 INJECT Ethanalamine 420 10 JE URE E xvwktzz TIHBO ITeZXBSgS cc CBc amp 2 9UbUWJvrzFrF Edit amp 20v 2d 2 EDC NHS Mix amp Inject Solution i Mis with NHS Fraction 2 af mis with solution Contact time s Flow rate pl rin OK Method rame Command Solution Contact Time s Flow Rate ulimin w p PRE CONC Specified in Immobilization Setup MISINJECT EDC MHS 50 50 D P WASH Ethanalamine b LIGAM
63. T Regeneration Scouting Experimental Parameters Regeneration parameters Flow rate 30 _ pl min Stabilization period 30 s Te Regeneration Scouting Results High viscosity solution Trend Chart Sensorarams Experimental design amp Sample Response Baseline Number of conditions 4 B Number of cycles for each condition 5 v Settings Condition Regeneration solution Contact time s il IalyHClpH3 0 30 2 Gly HCI pH2 0 30 3 Gly HCI pH1 5 30 4 50mM NaOH HE Display Sensorgram lstsample cycle Conditions i 2 Sensorgram Fc 4 x Application Wizard Assay Development Surface Performance 400 T Surface Performance Injection Parameters Sample Solution mia E JE X Value a Y Value 1 267 6 ia Binding stability Curve Name Fc 4 3 Contact time 60 ls Flow rate 30 pl rnin P Assay Step Purpose Sample P Cycle number Overlay Binding stability C Zoom Lock Regeneration 2 268 1 d LES kb d x09 uet attosta y taat 0 3 3 207
64. Pooling Yes OK Automatic Positioning Eject Rack Rack tray port 2wu27kb 4 B8BzxcCAUL OK Eject Rack Tray Rack Positions Next Te Kinetics Affinity Prepare Run Protocol Tahama n 1 Prepare Run Protocol Make sure the correct sensor chip is docked Make sure all samples amp reagents are loaded in the rack and microplate according tn the Rack Positions setup iVials should be sealed with rubber caps and microplate with adhesive foil Place the buffers on the left hand tray and insert the correct tubing s see below Mote Standby after run will use buffer A Make sure there is fresh water in the water bottle an the right hand tray If necessary empty the waste bottle before start af the run Estimated run time bh 47 min excluding conditional statements temperature changes and standby How Estimated buffer consumption Running buffer
65. Toolbar AJ Kinetics Affinity RI 0 RU Residual Residuals Y Residuals for a good fit Residuals for a poor fit RU Biacore T100 ao o 100 200 3200 400 sn A00 n 100 200 300 400 ins i ii i 5 119 Chi U value FIENDER Ce 672 862 ANS 18 15 25
66. 6 Toolbar Run Wizard Menu bar Run gt Wizard e Assay Kinetics Affinity Binding Analysis i Concentration A nalysiz Thermodynamics Assay Concentration Analysis New Methods and Templates 2 Open Browse Open Te Concentration Analysis Injection Sequence Detection Chip Capture SAMPLE 5 ample Enhancement REGENERATION 1 Regeneration 1 Biacore T100 5 125 Detection Flow path Chip Chip type
67. 54 54 IN S Kp Ka ka 5 5 IX Sau Ko ED 1 10 10 KORENA C 5 1nM 1uM 5 5 Ko187 5 2 855 2 86 Et 9c kK 0 Ze BI LR 30 ul min 2424 FS C RES 2 2
68. Save as Methods and Templates 7 Bia Users Don t Save Save in File nme Save Biacore T100 3 39 Standby flow 3 3 Ctrl Breakl Te Biacore T100 Control Software Immobilization of Prtein amp blr Ef iz Fie Edit view Run Tools Help zu X ProtreinA 20ug ml pH5 Lock scale NHS EDC 48000 D Ligand 3 Ethanolamine 44000 i 42000 1 cc 40000 38000 EDC NHS 36000 DH mmobiized 34000 32000 0 200 400 600 1400 1600 AbsResp LASD Slope RelResp Baseline Keywords in cycle 1 Value 5 paes 0 07 0 02 0 0 Baseline Chip CM5 5 350287 0 04 0 05 125 1 EDC NHS ContactT ime 420 5 374125 003 047 1503 3 Ligand FlowRate 10 5 374155 004 021 1513 3 Immobilized Ligand P
69. Keyword table C E3 2 Tools Keyword Table S Keyword Table Cycle Assay Step Purpose Sample Conc tuM Mw Da ites c E z Reset All Filters Add Keyword Rename Keyword D os i Warfarin d E Warfarin hii T des Warfarin i loka ea site M Concentration Unit Warfarin i Warfarin ui Y Naproxen Naproxen Naproxen Naproxen Naproxen Naproxen Naproxen Naproxen lt Naproxen Furosemide Furosemide Biacore T100 184 5 5 57 Menubar Toolbar Evaluation Explorer Work area Menubar Toolbar Evaluation Explorer W nsorgrom piot Baseline Binding level Binding stability m Report Point Table Binding to reference Work area Biacore T100 CER Sensorgram window AbsResp Baseline 73 50 RelResp Bqseline
70. Biacore T100 6 271 Eject Rack Rack tray port l OK Eject Rack Tray Rack Positions 9 Next T Thermodynamics Prepare Run Protocol Prepare Run Protocol Make sure the correct sensor chip is docked e Make sure all samples amp reagents are loaded in the rack and microplate according to the Rack Positions setup vials should be sealed with rubber caps and microplate with adhesive foil e Place the buffer s on the left hand tray and insert the correct tubing s see below Note Standby after run will use buffer A e Make sure there is fresh water in the water bottle on the right hand tray e fnecessary empty the waste bottle before start of the run Estimated run time 12h 13 min excluding conditional statements temperature changes and standby flow Estimated buffer consumption 3 3 aj Running buffer Approximately 200 ml 3 j 3 ge ue 7 Cc f Rn ee 7517 8
71. k k ORURO R RI RI Biacore T100 5 117 Residuals Y Residuals for a poor fit RU u Residuals for a good fit
72. lt Rr RU Rr Rmo R Biacore T100 78 5 fie 5 12 FS Kinetics Affinity Fit Kinetics Create Curve Fc 2 1 Ligand antibody 1 ug ml pH5 Sample antigeni Temperature 25 C Add Fit Current Fits Model 1 1 Binding v E j Description
73. DMSO 1 1 500 RU DMSO DMSO 10 RU 3 100 RU 10 000 RU DMSO 3000 RU DMSO Biacore T100 5 213
74. Minor Gridlines OK RU Adjusted sensorgram I Zoom Lock 350 E Lopta i m ad B 300 7 250 _ 200 amp e 150 1 E e 100 50 0 50 50 0 50 100 150 200 250 300 Time 0 baseline Biacore T100 9 305 9 5 QiBli amp z 9277 cU cte Excel Sensorgram window Copy Graph Biacore Word Pad Paint Bl Word Pad Sensorgram
75. Select Evaluation mode Single mode Next gt Biacore T100 206 5 Kinetics Affinity Select Data Create Curves Curve Fc 2 1 Ligand antibody 10ug ml pH5 Sample antigen Temperature ul min s s 6 Fc 2 1 30 7 Fc 2 1 30 8 Fc 2 1 30 9 Fc 2 1 30 10 Fc 2 1 30 Blank Subtracted Sensorgrams Zoom lock 0 Kinetics gt FS Kinetics Affinity Fit Kinetics Create Curve Fc 2 1 Ligand antibody 10ug ml pH5 Sample antigen Temperature 3 C Add Fit Model 111 Binding Parameters Cancel Biacore T100 5 207 Model 1 1 Binding Fit Kinetics Affinity Fit Kinetics Create Curve Fc 2 Ligand antibody 10ug ml pH Sample antigen Temperature 3 C Add Fit Current Fit kind 1 1 Binding E TT Bindin EM Wo Cycle 9 antigen 8 5 nM Quality Control Report Residuals Parameters Finish
76. SE SE DIBH 10 Biacore T100 5 77 RI RI RI 0 Constant 1 ka ka Rn
77. Control Sqmples Te Kinetics Affinity Control Samples Control sample definition Hun control samples Repeat control sample s every 11 sample cycles Control zamples MW Da Concentration Concentration Control sample id Biacore T100 218 5 Control Sample definition Run control Samples Repeat control Sample s every Control Samples Next gt 5 71 Excel 2 Excel 2 Excel t0 To Kinetics Atfinity System Preparations Frime before run Normalize detector Temperature settings Anal
78. Ilgnore Biacore T100 5 61 BXE 5 2 Excel Excel Excel t Te Kinetics Affinity System Preparations Frime before run Normalize detector Temperature settings Analysis temperature C Sample compartment temperature C Prime Normalize Temperature settings Analysis temperature 25 C Sample compartment temperature 25 C Cycle Run List Te Kinetics Affinity Cycle run list 2 Startup buffer 3 Startup buffer 4 Startup buffer 5 Sample antigeni D 11500 mm Sample antigeni D 11500 Sample antigeni 1 1 11500 8 Sample antigeni 2 2 11500 9 Sample antigeni 4 3 11500 10 Sample antigeni 8 5 11500 m Sample antigeni 17 11500 12 Sample antig
79. Include Next gt Biacore T100 5 89 Kinetics Affinity Select Data Create Curves Curve Fc 4 3cor Ligand N A Sample Furosemide Temperature 25 LC ER Lonc Flow Contact Time Diss Time ndis ads pM l min s s 47 Fc 4 3 corr 3 48 Fc 4 3 corr 3 48 Fec 4 3 corr 3 50 Fe 4 3 corr 30 51 Fe 4 3 corr 30 52 i Fe 4 3 corr 30 53 Fe 4 3 corr 30 Blank Subtracted Sensorgrams Zoom lock T eme a nenne i z Affinity gt ES Kinetics Affinity Select Affinity Data Create Lurve Fc 4 3cor Ligand N A Sample Furosemide Temperature 25 C Settings Calculate response at position 4 seconds before injection stop with window 5 seconds 4e 585 5 Concentration Biacore T100 90 5 GU Req RU Rea Next gt ES Kinetics 7 Affinity Fit Af
80. pH Preconcentration binding is too slow pH Biacore T100 4 45 4 Bin amp 43 Td 2e
81. 60 Qe a5 B S B 8Bila 2 FRP CRUS VN i EEBOIBNICIAU x o o iB
82. AEE RU 10 Carry Over co binding co_boseline Startup Binding to reference Stability baseline Biacore T100 11 42339 5 12 42338 9 13 42338 5 14 42337 9 15 42337 3 16 42336 8 17 4233
83. Reuse Chip Chip type 7 Insert Chip New chip O Reuse chip New chip mwe og v Chip id 080415 0213 12114 Chip lot no optional 10113353 Chip id amp Chip lot no optiona Biacore T100 6 1 ILLAS s r AUS e D Insert Chio Dock Chip Biacore T100 Control Software eo Inserting chip 1 32 Dock Standby flow Standby flow A 4 65 ml 24 3
84. Conditioning Solution FAERIT Id GRO SE contact time s Biacore T100 5 59 Number of injections Startup Solution Number of cycles 3 Next gt Ta Kinetics Affinity Injection Parameters Sample Contact time 120 s Elow rate iuMminl Dissociation time 120 s 7 Extra wash after injection with mm Regeneration Solution Gl HClpH25 7 High viscosity solution Contact time s Elow rate l min Stabilization period s Sample contact time 120s Flow rate 30 ul min Dissociation time FE MEISIE 120 Regeneration Solution High viscosity solution 40 contact time 30s Flow rate 30 ul min Stabilization period Os Next gt
85. Rrz Fit Fitlocal 0 RI RI Fit Constant Initial value 0 es Parameter Setting OK Fit Biacore T100 122 5 XE 5 29 Current Fits Current Fits 1 1 1 Binding Description Two State Reaction Delete Biacore 100 Evaluation k J Are you sure vou want to delete this Fit OK Curent Fits 1 Two State Reaction Description Biacore
86. Biacore T100 Evaluation Software ERA PA PAN om Run gt Stop Run Hiarnre T100 AN This will stop Ehe run stop Run Run Stopped Finishing curent cycle please wait Abort cycle hn Ctri Break Ctrl Breakl Biacore T100 Evaluation Software Biacore T100 66 5 5 1 2 Evaludtion Biacore T100 Evaluation Software Kinetics antigen antibody blr tet ndow Help Concentration Analysis v Thermodynamics Eak m Curve Name Fc 2 1 e
87. Start Desorb Standby flow 3 4 Prime 3 Biacore T100 278 7 7 1 2 Desorb and Sanitize Jd NCOZ U zAzZAOwWWB RddO 389345 d2 1 1 1 20 C 20 C A B C D A J B C D Biacore Maintenance Kit type 2 BlAdesorb solution 1 0 5 96 SDS BIAdesorb solution 2 50 mM Gly NaOH pH 9 5 BlAdisinfectant solution IRW 6 ml 80 ml Maintenance Tools Desorb and Sanitize Start Desorb and Sanitize This procedure removes adsorbed material and disinfects the tow system The procedure iz divided into five steps Total run time is about one hour Followed bu a recommended standby time of 3 4 haurs NOTE Use the Maintenance Chip for this
88. fio Sp m UL CJ ESAE CZ BS S Biacore T100 162 5 Eject Rack Rack tray port l OK Eject Rack Tray Rack Positions Next gt T Method Builder Prepare Run Protocol Prepare Run Protocol Make sure the correct sensor chip is docked Make sure all samples amp reagents are loaded in the rack and microplate according to the Rack Positions setup yials should be sealed with rubber caps and microplate with adhesive foil Place the buffer s on the left hand tray and insert the correct tubing s see below Note Standby after run will use buffer A Make sure there is fresh water in the water bottle on the right hand tray If necessary empty the waste bottle before start of the run Estimated run time 1 h 14 min excluding conditional statements temperature changes and standby flow rna D j ECKBUVGCIERESEIR WERA D3ERERL LZ EBDRSIJGZnctiu Start Save as Methods and Templates
89. Sensor Chip Maintenance Menu bar Tools More Tools Tools 1 Maintenance Tools esorb Desorb and Sanitize Empty Buffer Tubing Jl Normalize wash Buffer Tubing S E Test Tools m System Check A Service Tools Software Problem Report ad Flow System Wash aj Superclean This procedure remaves adsorbed material From Ehe Flow system Total run time is about 20 minutes Do nat run this procedure below 20 75 NOTE Use the Maintenance Chip For this procedure The surface on other sensor chips may be damaged by the solutions used Last run time 5IZHIEHHB 9 25 AM Biacore T100 7 275 Biqcore Maintenance Kit type 2 BR 1006 51 BIAdesorb solution 1 95ml x2 BlAdesorb solution 2 95ml x2 BIAtest solution 65 ml BlAdisinfectant solution conc 10mlx3 BlAnormalizing solution 90 ml HBS N Buffer 10 X 50 ml Sensor Chip Maintenance 1
90. Immobilization pH Scouting pH t pH 5 200 ug ml 10 mM Immobilization pH Scouting Tik U 7j 7 FIKIA 27 274518 ET 50 mM NaOH Biacore T100 28 3 Toolbar Run Wizard Menu bar Run gt Wizard Do ee Run Wizard emplate mx Surface Preparation m Immobilization pH 5cnulinn SS
91. Ko 1 10 10 cCSBEXUE E ug m Biacore T100 46 4
92. wait Commands Regeneration Remove Startup Biacore T100 5 99 T Method Builder Main Cycle types Description of selected cycle type This cycle is used in start up steps General Settings d Contains injection sample and regeneration The sample is of the type low sample consumption not single cycle kinetics to save time during start up procedures The normal sample cycle could be used Assay Steps as a start up cycle also Cycle Types I gt Variable Settings Commands Report Points Verification N v Setting for Sample 1 Low sample consumption ww Method Variables Evaluation Variables Sample solution Is variable Set Hope as variable Dissociation time s ANE EE s Flow rate ul min Flow path Predip CI Mis with Fractio lution action o 72 of mix so Stabilization period after mix s Type Low Sample consumption contact time s 1 Dissocia
93. InjectAndRecover 5 8 General sample Dual Inject Dual Inject 1 lt 2 Genergl If Then Method Variables Evaluation Variables 2 Method Warables E valuation Variables Method Variables Evaluation Variables Set property as variable Evaluation purpaze Solution Kinetics Affinity v Contact time 2 Dissociation time s Flow rate pl min Extra wash solution Fredefined variables Value type Mumeric Numeric User defined variables Value type Biacore T100
94. Select Evaluation mode 1 Single mode Batch mode Bath mode 5 14 Sample Show average blank s Include Biacore T100 5 69 ES Kinetics Affinity Select Curves Create Select evaluation made Single mode Batch made Cures Curve Ligand antbodylDus my Sample aoaie nae a Qe a o rn nM pl min s s 1 1 30 2g 30 4 3 3 3 30 30 Z
95. BSA 10 mM 10 mM Borate 1 M NaCI pH 8 5 Biacore T100 26 3 5 200 ug ml 10 mM pH 0 5 2 pH pH 3 5 27 Immobilization pH Scouting 10 mM pH SA 100 ug ml 10 mM Borate 1 M NaCl pH 8 5
96. EN RE View Base Line F9 0 Biacore T100 52 4 4 4 Application Wizard Assay Development Regeneration Scouting 5
97. Quality Control 7 5 26 5 26 Quality Control 5 O Q Quality Control Rieport Residuals Parameters o Kinetic constants are within instrument specifications o Kinetic constants appear In be uniquely determined o No significant bulk contributions RT Faund o Check that sensorgrams have sufficient curvature 9 amp o Examine the residual plot Pay attention to systematic and non random deviations ka 103 107 1 Ms kg 105 0 5 1 s 7 ks k Rmax
98. Biacore T100 58 5 Detection Flow path 2 1 4 3 Chip Chip type Capture 2 4 Sample Regeneration 1 2 Carry Over Next gt Te Kinetics hffinity etup Conditioning Bun conditioning cycle Contact time mum s Mumher of injections Startup Run startup cycles Solution Number of cycles Solvent correction Bun solvent correction Number of injections Repeat after sample cycles
99. Setup Run 3 Define all values at run time Define all values in method JFRiBIE E CAJJSg lEEKUIS XV v F SRSRIETZ 7 Z7UL HFecUcthe Define some values in method and others at run time 5 273 OO Bs c oll E CAN7 U Of ootSSIBSRIXE PROS BU IC 73 Verification Biacore T100 6 267 T Method Builder Main Verification results The method has been verified and can be used to setup a run General Settings Assay Steps Cycle Types Variable Settings Verification SetupRun The method has been verified and can be used to set up arun Setup Run Te Method Builder Detection Detection Flow path
100. Menu Export Positions Menu Simple Position Import Biacore T100 5 63 5 4 Control Sample R1A1 R1A12 12 Ta 060327 Thermo Rack Positio Rea ent Rack 2 Positinn uc Content Sample 1 j 148 Negative control Control sample 148 Negative control Control sample e e 148 Negative control Control sample i 148 Negative control Control sample 148 Negative control Control sample ee i 148 Positive control Control sample F G i 148 Positive control Control sample 96 Deep Well Microplate i 148 Positive control Control sample
101. 269 T Method Builder Cycle run list Assay step name Sample 1 Solution Sample 1 Conc nM Sample 1 MW Da buffer buffer buffer negative control positive control sample 1 sample 1 sample 1 sample 1 sample 1 sample 1 sample 1 sample 1 sample 1 sample 1 negative control positive control sample 2 sample 2 sample 2 sample 2 sample 2 sample 2 v Next gt Biacore T100 270 6 6 3 T ADITU eT FERT AE System Preparations Prime before run Normalize detector Temperature settings Analysis temperature C Sample compartment temperature C Prime Normalize Next gt ie XE To Method Builder Rack Positions mina mmm Control sample 25 88 negative control Control sample 88 negative control Control sample 88 negative control Control sample 88 positive control Control sample 88 positive control Control sample 88 positive control Control sample 88 positive control Control sample 88 sample 1 Sample
102. Evaluation 9 1 Start BIAprograms Biacore T100 Evaluation Software Biacore T100 Evaluation Software ir z Sensorgrarn Plo au Bar Char AJK Evaluation Explorer n Biacore T100 9 293 9 2 Cg File gt Open GB C BIA users C BIA users T100 demo File Binding Analysis OK Biacore T100 Evaluation Software KineticsAffinity 1 1 interaction blr LS k AJ Kinetics Affinity Concentration Analysis Thermodynamics F a Sensorg am Plot Ba Chart Evaluation Explorer Evaluation Explorer H F All sensorgrams 7E z Curve Name Fc 2 1 J Assay Step Purpose D verlay me Cycle Overlay ensorgram j All sensorgrams RU Sensorgram E Plot Baseline Sample Binding level Binding stability Binding to reference Report Point Table rj R
103. Evaluation Biacore T100 Evaluation Software C Documents and SettingsbiacoreWDesktopvl100ManualMCSK CK_antibdy antigen bme E Ef Plot as Bar Chart Curve Names Overlay e Sensorgram aJ All sensorgrams RU 5 Plot 37100 C Zoom Lock Baseline Sample Binding level Binding stability Binding to reference Report Point Table rj Report Point Table Kinetics Affinity AJ antigen 1000 1200 1400 1500 s HE 5 22 Keyword table C E3 2 Tools Keyword Table Keyword Table Cycle Assay step purpose Sample Conc nM Mw Da M v v v Reset All Filters 1 Conditioning 2 Startup buffer 3 Startup buffer 4 Startup buffer 5 Sample antigen B Sample f antigen j 7 Sample antigen 8 Sample antigen1 8 Sample antigenl 10 Sample antigen 11 E Sample antigenl 12 Sample antigen 1 E Sample antigen2 1 4 Sample antigen M 15 Sample antigen2 16 Sample antigen2 17 Sample antigen2 18 Sample antigen2 1 g Sam
104. Finol Bound Biacore T100 3 41 3 1 3 Aim for Immobilized level 3 2 Toolbar Run Wizard Menu bar Run gt Wizard Do te Run Wizard Template pope E Les H Immobilization amp s SSal Development Regeneration Scouting Buffer Scouting Surface Perfarmance Eg Co ntral Experiments Kinetics Linked Haachinns Kinetics Mass Transfer E Ass Concentration Analysis Binding Analysis Thermodynamics Surface Preparation Immobilization New Methods and Template
105. XD coUv duUlcla Hts g WSJAZSSUAS sSLL CES rine nad NN Cle b Ux TOSS vm I i D 9 Di On o m EILAN ORAAL QU 5 35 91 5 3U 977 0u 3 JALAN OHE HITA Ab IC
106. 733 8 f81E 100 Evaluation Add Plot i LINE xl Plot Settings Plot name Plot Plot type e Feport Point vs ariable C Report Point vs Report Point xis setting Y xis A Axis Report Point stability lt Variable Cycle number lt Response Type Relative response gt Hee Einish Cancel Plot name MW correction Plot type Report Point vs Variable Axis setting Y Report Point binding Response type MW adjustedresponse X Variable Cycle number D LIMEN xl Plot Settings Plot name MW correction Plot type e Report Point vs Variable C Report Point vs Report Point r Axis setting Y Axis X Axis Report Point binding m Variable Cycle number Response Type Mw adjusted response Hee Finish Cancel Finish Biacore T100 5 231 W Evaluation Explorer azl Edit x Remove 7 Sensoreram i sJ All sensorerams p Plot m a Baseline Sample me Binding level m a Binding stability a Binding to re
107. Toolbar Sensorgram window Report point table Event log Status bor Temperature Sample compartment temperature Run Biacore T100 1 5 1 2 1 2 1 Insert Chip T100 Insert Chip e Mew chip Q Reuse chip Mew chip Chip id 80415 02193 12114 Insert Chip Mew chip Q Reuse chip New chip Chip type Chip id Chip lat na Series S CM5 New Chip
108. Biacore T100 50 4 Regeneration command LA Menu bar Commands gt Regeneration Regeneration Vial well position R2B2 amp Contact time s High viscosity solution Minimum required volume in ral well For this injection 72 pl 530 605 OK Cancel Te Biacore T100 Control Software regeneration check blr i File Edit View Commands Run Tools Help A Em B c e Cycle 1 a Curve Sensorgram Fc 4 I 9131 Bh i mE C Lock scale 100 200 500 Fc Time Window AbsResp SD LASD Slope RelResp Baseline Id Keywords in cycle 1 Value 4 1050 SEE STER UT 0 05 0 05 LU Yes baseline 1 4 2310 5 3897072 28 69 018 1534 5 No binding 1 4 2450 5 33 857 388 0 21 207 0 No stability_1 Flow 30 Flow Path 3 4 Online COM1 Temperature 25 00 9C Running manual run Sample compartment temperature current 25 C sek 25 C Run time 9 min
109. Blank Subtracted Sensorgrams E Zoom lock Remove Selection Biacore T100 5 71 pa Kinetics Affinity Fit Kinetics Create Curve Fc 2 Ligand antibody 10ug mlpH5 Sample antigen Add Fit Temperature 5 C Z Cancel Model x 1 1 Binding Madek 1 1Bindinn T BN Bivalent Analyte Heterogeneous Analyte Heterogeneous Ligand Two State Reaction RU Fit Biacore T100 72 5 5 9 B A 1 1 Binding A B AB 1 Bivalent Analyte A B AB AB B AB2 2 2 AB
110. CM NHS N NHS 3S S S S Biacore T100 24 3 m SEU
111. Yes OK Automatic Positioning Biacore T100 5 221 Eject Rack Rack tray port OK Eject Rack Tray Rack Positions Next gt Te drug Prepare Run Protocol Tahoma Prepare Run Protocol Make sure the correct sensor chip is docked Make sure all samples amp reagents are loaded in the rack and microplate according tn the Rack Positions setup ials should be sealed with rubber caps and microplate with adhesive foil Flaca the buffers on the left hand tray and insert the correct tubing s see below Mote Standby after run will use buffer A Make sure there is fresh water in the water bottle nh the right hand tray If necessary empty the waste bottle before start of the run Estimated run time 19h mm excluding conditional statements temperature changes and standby How Estimated buffer consumption a Running buffer j j Approximately
112. Biacore T100 20 2 2 1 2 RU 2 3 Fe Time Window AbsResp SD LASD Slope RelResp Baselne ld 1 1320 5 368816 008 010 000 00 Yes baseline 1 1 1970 5 5965026 258 023 138 227210 No binding 1 1 2120 5 368737 015 014 005 1 9 No stability 1 Id baseline 1 10 binding 1 5 stability 1 3 10 binding 1 Injed Ready 20 pl de h3 e e baseline 1 RU o CODD RU RelResp 0 0 binding 1 stability 1 RelResp baseline 1 RU
113. pH4 pH NHS NHS pH8 5 pH pH pH5 Immobilization pH Scouting RU 100 ug ml Immobilization pH Scouting Biacore T100 34 3 3 1 2 Toolbar Run Wizard Menu bar Run gt Wizard Do Te Run Wizard Template C Surface Preparation Immobilization pH Scouting 2 Assay Development vi Regeneration Scouting Buffer Scouting Surface Performance C Control Experiments
114. 305 ep cM m lid 305 y b ed x AAO EES 289 c DX RN ans pde DA IM IM MI M M M AT 290 Biacore T100 nis oo PH 143 193 NIS NE uS IUE IM MIL E 24 53 93 123 125 136 259 260 Sh a D C LI LM LM UM E LL EE 301 302 E rH Em 12 13 A T EA E i GE 68 88 P 212 213 223 229 Vd TE aris PN E 71 72 77 78 114 115 119 120 TEE EE P MN TU 24 41 45 53 54 55 85 93 193 194 eA TEST iz E s DEAL urs Ly BET EE 85 Rc 194 210 Re M JAN uuu ML MISMA eT 55 zz dn er E E T T T NN T T 27 45 124 190 211 233 or cor WINNIE 76 119 B ATAO ee oo 307 pe DER e LIA EMI LEE UE LI UAE E EM EE 173 207 LL 71 73 74 76 77 114 116 117 118 119 123 188 E a eut 7E pU C u 76 118 EAE der PASO Ur dba m E 248 IIIRO 187 230 231 SEN TTE 55 92 SE IN ENT aaa EEEE T OE EEE 87 92 c Lekkf V V T V LZLEE4 _ 24 144 145 169 kk ks 15 45 233 239 I TA 53 54 93 205 23
115. 65 ml 24 4 Status bar 8 2 7 Toolbar Eject xp Menu bar Tools Eject Chip Biacore T100 AN This will eject Ehe sensor chip Eject chin Eject Chip Biacore T100 control software Biacore T100 Biacore T100 290 8 3 8 3 2
116. Walug Valule Tos Biacore T100 186 5 Calor By Sort Curve Fitting Adjustment For controls Ranking Table Columns ME Ranking amp 2 J w 2d E Ranking Ranking Settings O Mane One Ranking Boundary Two Ranking Boundaries Ranking Boundaries First Value Second Value RU RU One Ranking Boundary 1 First value 2 RU Two Ranking Boundaries 2 First value Second value 50 RU Finish Fi
117. DMSO 596 DMSO DMSO 10 0 05 Surfactant P20 s Big 5 69 PBS 10X 1x1000 ml BR 1006 72 pH 0 1M phosphate Buffer 27 mM KCI 1 37 M NaCl 1018959 V DMSO 0 01 M phosphate Buffer 2 7 mM KCI 0 137 M NaCl 596 DMSO pH7 4 DMSO DMSO 0 22 um DMSO UV spectrometry DMSO pH DMSO
118. Next gt T System Check Rack Positions 695 BlAtest solution Empty Empty vial with cap min capacity 400pl Empty Empty vial with cap min capacity 200pl Empty Empty vial with cap min capacity 100pl Empty Empty vial with cap min capacity 100jl BIAtest Solution 1 5 ml 695 uu 1 5 m 4 L Start Save in e Bia Users v Q 2 e mn Methods and Templates My Rec Documents e File name KineticsAffinity 1 1 interaction bme L MERE My Network Save as type Biacore T100 Evaluation Files bme v File name ADULT Save BC D Empty Buffer Tubing Biacore T100 288 7 Results System Check System Check Date Biacore T100 Control Software Version 1 0 12113 Configuration IFC105 D05 Sep 2005 Evaluation Date 05 Sep 2005 25 0 C 050905 2086 26
119. 255 6 1 QI Startup 1 Startup Thermodynamics 5 times as entered A Control sample 1 Control sample Thermodynamics 1 time as entered Thermo 1 sample Thermodynamics 1 time as entered Startup 1 Control Sample 1 Thermo 1 Recurrence Control Sample Control Sample 1 Hecurence Repeat assay step within 9 Every C2 Distribute Run assay step ance fir Number af replicates Repeat assay step within
120. 1 1 20 20 Sample compartment 20 CME Biacore Maintenance Kit type 2 BlAdesorb solution 1 0 5 96 SDS BIAdesorb solution 2 50 mM Gly NaOH pH 9 5 A Tools gt More Tools gt Maintenance Tools Desorb Start This procedure removes adsorbed material from the Flow system Total run time is about 20 minutes NOTE Use the Maintenance Chip For this procedure The surface on ether sensar chips may be damaged by the solutions used Do nat run this procedure below 20 C Next gt Desorh Required zalutions fram Maintenance Ft Bl amp desorb solution 1 Bl amp desorb solution 2 Next gt BlAdesorb solution 1 BlAdesorb solution 2 16 mm 15 mm 1650 pl
121. 0 8 ml x24 Sample and reagent rack 16 mm 4ml x9 15mm 4 ml x94 15m 11 mm x 24 7 mm 0 8 ml x45 Rack tray 96 well 384 well x1 Rubber caps type 3 Rubber caps type 2 Rubber caps type 5 BR 1005 02 amp BR 1004 11 J BR 1006 55 EN 7 mm Plastic Vials 1 5 ml Plastic Vials 16 mm Glass Vials 15 mm Plastic Vials BR 1002 12 BR 1002 8 BR 1002 09 BR 1006 54 lt Biacore T100 1 13 1 11 Reagent rack EI ABC 123 A1 A2 A BC D EF G Biacore T100 14 2 2 3 Toolbar 12
122. 5 Buffer settings After run mec BHs ND Assay Steps Biacore T100 5 237 T Method Builder Overview Surface conditioning General Settings Conditioning Conditioning 20 times as entered A Assay Steps A C Inject and Recover Sample Inject and Recover 1 time as entered Variable Settings Cycle Types Cycle Run List Assay step properties Base settings Recurrence Name Surface conditioning Repeat assay step within Purpose Conditioning v Every cycle dur Conditioning v Distribute occurences evenly Run assay step once first Run assay step once last Assay step preparations 5er of replicates Temperature 3 times Buffer As entered 1 2 3 1 2 3 O Order 11223 3 Random Number of replicates Times 3 Injection and Recover Biacore T100 238 5 T Method Builder Main Overview Surface conditioning Genera
123. D 5 19 Menu Export Positions DEFALAS Menu Simple Position Import Biacore T100 5 20 S Control Sample R1A1 Te 060327 Thermo R
124. Y Adjustment Report point response 0 baseline de Yr Adjustment i i Report Point response 0 Injection Event response 0 OK RU Adjusted sensorgram 30 amp binding binding amp isimatimm Response 0 baseline a iutabi ivtabi lity Aty abiity x ability binding s binding Ebaseline e amp binding stability 50 50 100 150 200 250 300 Time 0 baseline s 9 3 4 RU Adjusted sensorgram 450 350 M en e Response 0 baseline m e 100 0 100 200 300 400 500 600 Time 0 baseline s Biacore T100 300 9 RU Adjusted sensorgram ic binding ability ding ability gS ty S nding binding Ebindin amp sability stability Erie Response 0 baseline tbinding bi 100 50 50 100 150 200 250 300 350 9 3 5
125. Overview 6 Biacore T100 5 235 T Method Builder Main Concentration unit ng ml General Settings RM Data collection rate 1Hz Sample compartment temperature 25 C Detection Multi Inject and Recover Sample Inject and Recover 1 time as entered Settings for assay step Surface conditioning T Variable Settings BandAl He sw saeas General settings Biacore T100 236 5 T Method Builder Main Data collection rate Detection Sample compartment temperature Hz C Vary with analysis temperature Concentration unit uffer settings u m Y Miscellaneous Running buffer After run 5 C Specify analysis temperature after run D Data Collection rate 1Hz 2 Detection Multi 1 2 3 4 Sample compartment temperature 4 45 C 25C Concentration unit
126. Rack Positions Next gt 9 Te Method Builder Prepare Run Protocol Tahoma a 10 Prepare Run Protocol Make sure the correct sensor chip is docked Make sure all samples amp reagents are loaded in the rack and microplate according tn the Rack Positions setup ials should be sealed with rubber caps and microplate with adhesive foil Place the buffers on the left hand tray and insert the correct tubing s see below Mote Standby after run will use buffer A Make sure there is fresh water in the water bottle nh the right hand tray If necessary empty the waste bottle before start of the run Estimated run time 55 min excluding conditional statements temperature changes and standby How Estimated buffer consumption ad Running buffer j j i Approsimately 100 ml Start Save as Methods and Templates 7 Bia Users Don t Save EG Methods and Templates My Recent LJnakayama Docu
127. Sensoreram All sensorgrams gm Plot A al Baseline Sample a Binding stability Controls stability Concentration Analvzis m LA Concentration Analysis Evaqluation Explorer qpic Concentration Analysis Biacore T100 5 139 5 36 RU Biotin 1600 1400 1200 Exclude Curve 1000 Exclude Cycle Show Sensorgram Relative Response HI Labels 600 Scale Copy Graph anti Export Curves i Gridlines 200 Legend 20 zt 4 Bi at 100 120 Concentration nimi Exclude Cycle Calibration Table Response Calc Conc CY RU ng ml 9o MA
128. ABCD 70 10 mD Start i 2 3 Biacore T100 282 7 Empty Buffer Tubing Step 3 Remove the tubes fram the ethanol battle and allow them ta hang in the air This step empties the buffer selector of liquid stat ABCD 70 Start Empty Buffer Tubing The Empty Buffer Tubing procedure iz completed Close Close BC D E Biacore T100 7 283 7 1 4 Wash Buffer Tubing ABCID BSA 30 Biaco
129. Biacore T100 5 147 S Viscosity relative to water at 20C 20C Use standard value 1 00 1 O Enter value cenrars Convert diffusion coefficient from temperature T to 20 C Temperature T 25 CE D at temperature T 4 00e 11 imos CONUERT Diffusion coefficient at 20 C 3 49e 11 MKA 20C 20 Biacore T100 148 5 5 4 1 Toolbar Run Method S Menu bar Run Method 2s Te Open New Method in EEEE Templates Modified Biacore Methods Biacore Methods Open
130. Capture Sample Enhancement 2 Regeneration 1 2 Next gt Te Concentration Analysis Setup Conditioning Bun conditioning cycle Contact time s Number of injections Startup Run startup cycles Mumber of cycles Conditioning Je Solution contact time s Number of injections Startup Biacore T100 126 5 Solution Number of cycles Next gt 3 Te Concentration Analysis Injection Parameters Sample Conta
131. LM Biacore T100 Control Software Fie view Run als Help Te e iL nw P Bl Manual run 4 Application wizards BRL CODRRFHZ 4 U FN pH c Methods Manual run Biacore T100 2 15 2 1 Toolbar Start Manual run Ep Menu bar Run Manual run
132. 262 6 Method Variables Solution Evaluation Variables Evaluation purpose JG L TRZ ID SEE Evaluation software _ User defined variables F Add Evaluation purpose I amp Sample Evaluation purpose 7 Kinetics Affinity Thermodynamics Concentration Affinity in solution Kinetics
133. 5 57 5 1 1 Toolbar Run Wizard Menu bar Run gt Wizard Do Te O0pen7New Wizard Template C Surface Preparation Immobilization pH Scouting Immobilization CI Assay Development Regeneration Scouting H 070814 V Buffer Scouting 070313 Surface Performance 1080314 2 Control Experiments C0805 Kinetics Linked Reactions Kinetics Mass Transfer E Assay 2 Binding amp nalysis Concentration Analysis Thermodynamics Look in E Methods nd Templates Name Assay Kinetics Affinity New Methods and Templates 2 Open Browse Open Te KinetirsrAffinity Injection Sequence Detection Chip Elow path Chip type Capture REGENERATION 1 Regeneration Cary Over 1
134. Heterogeneous Analyte A1 B A1B A2 4 B A2B 1 2 Heterogeneous Ligand A B1 AB1 A B2 AB2 2 Two state Reaction A B AB AB 1 antigen m fx Curre Fc 4 3 Ligand antibody Sample antigen Temperature 25 C Fite 1 1 1 Binding E c 1 i ti A mm C Quality Control Report Residuals Parameters o Kinetic constants are within instrument specificatians o Kinetic constants appear In be uniquely determined o Mo significant bulk contributions RT found o Check that sgnsnrnrams have sufficient curvature o Examine the residual plat Pay attention to systematic and non random deviations Biacore T100 116 5 1 1 binding
135. Biacore T100 Evaluation Software DMSO DMSO 1 x y NO0 lt D DMSO 596 DMSO DMSO DMSO 1x PBS no DMSO 4 69 DMSO 4 DMSO 1x 96
136. Rack Positions Next gt l Te Method Builder Prepare Kun Protocol Prepare Run Protocol Make sure the correct sensor chip is docked Make sure all samples amp reagents are loaded in the rack and microplate according to the Rack Positions setup viales should be sealed with rubber caps and microplate with adhesive foil Flace the buffer s on tha left hand tray and insert the correct tubing s see below Mote Standby after run will use buffer A Make sure there is fresh water in the water bottle nn the right hand tray If necessary empty the waste bottle before start of the run Estimated run time 1 h 38 min escluding conditional statements temperature changes and standby Flow Estimated buffer consumption J z f Buffer amp Approximately 100 ml E Start Save as Methods and Templates 7 Bia Users Don t Save Biacore T100 5
137. 5 36 5 37 Cs Biacore T100 138 5 Concentration Analysis Create Samples E Concentration Analysis Create Calibration Control Samples Samples Sample T able Sample Plot Biotin Response Calc Conc RU Cycle Sample Id RU Ing mij 1200 12 501 2436 01 13 501 2436 01 14 501 2436 01 15 501 2436 01 Avg 16 501 2436 02 19 501 2436 02 Avg 33 63 20 501 2437 01 18 8 21 501 2437 01 Avg 18 67 Relative Response e e e 22 501 2438 01 f 23 01 23 501 2438 01 Avg 26 501 2439 01 27 501 2439 01 15 20 25 3 35 Measured Concentration naml BIB Zr CD Sample Table Dil Fact AREE Calc Conc CV 1873 CV 5 36 Finish Evaluation Explorer
138. Biacore Biacore T100 http www biacore com diffusion calculator E FRIHT E GC E Biacore T100 Diffusion Coefficient Calculator Converter For users af Biacore T100 sofware version 2 0 or later This on line toolis designed ta help you calculate difusion coeficients far use in Calibratian Free Concentration Analysis assays TO access this section vou need to be logged in anc have a validi Product Key registered In vour account details If vou do hot have a Blacore website account vet you can signup hera FORGOT FHzzUuloaED F Lol User ID Password LOGIN Biacore T100 146 5 Biacore T100 Diffusion Coefficient Calculator Converter For users af Biacare T100 software version 2 0 ar later This on line toolis designed tn help vau calculate difusion coeficients far use in Calibration Free Concentration Analysis assays Calculate diffusion coefficient at 20 C I I I I l I Molecular weight 1 s0000 Da I j Frictional ratio 99 9 Choose molecular shape gt Globular 1 2 en I I Enter value I I I l viscosity relative 8 Use
139. E 5 11 RI 0 RU Residual Residuals Y Residuals for a good fit Residuals for a poor fit Response 100 100 z200 300 400 sgg 100 1 z200 300 400 Time Z Time Z Chi 6 U value 15 25 SE Standard error Parameters
140. Tools Keyword Table Keyword Table E Oycle Assay Step Purpose Sample Conc uM MW Dal ites E z Reset All Filters buffer i buffer 0 mu buffer 0 i Add Keyword H Warfarin D j Rename Keyword Warfarin 100 t A B NR Remove Keyword Warfarin 25 1 _ Warfarin 125 Marfarin 625 i Warfarin 3125 Warfarin 15625 Warfarin 078125 entation Warfarin 125 E xp 50 25 GEC Naproxen _ l 625 1 i Naproxen 3125 Naproxen 15625 i Naproxen i 0 78125 Naproxen 125 eese s ji Furosemide 20 Furosemide 10 Biacore T100 5 225 Menubar Toolbar Evaluation Explorer Work area Menubar Toolbar Evaluation Explorer ogram ot Baseline AbsResp Binding level Baseline RelResp Binding stability Bseline RelResp Binding to reference
141. Cycle Curve Ligand Sample Dilution factor Flow hl min Initial rate RU s 10 5 QC ratio QC Temp C MW Da 7 3 88 D m2 s Blank used Expand all cycles Biacore T100 168 5 Use reference subtraction data l ideda 1 Calibration free concentration Included blanks Zoom lock Ap ON TS Un ij m nM PU ii
142. Do Manual Run Flow g Flow rate ER min Flow path Reference Detection in How cells 1 2 3 4 subtraction Q E Flaw path 1 O E Flow path 1 2 D B Flow path 2 Q EI Flow path 3 4 12 oB Flow path 3 e Flow path 1 2 3 4 01211022 Q a Flow path 4 IOOOOOOCO Flow rate 1100 ul min Flow path Rack Start _ Start 2 1 l min s ub 28 ul
143. No n Biacore T100 156 5 Te Method Builder Main Assay steps Define variable handling for each amp ssay Step General Setting s Define all values at run time Assay Steps N O Define all values in method Define some values in method and others at run time Cycle Types Variable Settings 7 I M Values for these variables will be defined at run time Command Variable D 20 C Blank SeupRun Dilution Assay Steps Blank Define some values in Method and others at run time Flow rate hl min Setup Run Sample solution Buffer ADe MW Da RAJ D 20 C Blank Yes Dilution RAD Verification Biacore T100 5 157 T Method Builder Main Overview Verification results General Settings Assay Steps
144. Biacore T100 16 2 save Results From Run s Savein T100manual v Qd e E My Recent Documents hy Computer My Network Saveastype Result file bl v C Biq Users Sae T Biacore T100 Control Software manual blr bz File n iew Commands Run Tools Help i kd x A r Epli cyde 1 m Curve Sensorgram Fc 1 AIL ZIZiGIB 35940 EPAR M 0 5 10 15 20 40 C Lock scale Keywords in cycle 1 Flow 30 Flow Path 1 Online COM1 Temperature 25 00 9C Running manual run Sample compartment temperature current 25 C set 25 C Run Lime 1 min Biacore T100 2 17 E 2 2 Biacore T100 18 2 2 1 1 Inject command
145. NHS pH 8 5 DMSO Instrument handbook Biacore T100 3 27 3 1 1 pH CM5 CM CM 05 pH o Ou HOMME dic pH A
146. CON COO COO CONHCH CH OH NHS BR 1000 50 EDC N ethyl N 3 dimethylaminopropy carbodiimide hydrochloride NHS N hydroxysuccinimide 1 M ethanolamine hydrochloride pH 8 5 EDC NHS 10 ml 200 ul 7 mm 20 1 4 C 200 yl 1
147. Y D xf km 0 98 X 0 3 x h x w x D fo h w XR a Biacore T100 5 145 5 41 CFCA 20C 5324 5x10 11 fxmreixMu173 m s f FRE N rel 20C Mw Do D Biacore Biacore T100
148. Binding level Work area Evaluation Explorer Biacore T100 226 5 Evaluation Add Solvent correction E Solvent Correction x zx PER ien Zoom lock E 9 T x 5 i S i E trapalation Range Report Paint Hange 1500 1000 500 s00 1000 1500 Mone Extrapolate ly Shaw repart paint range uis g Response Ref zn OK di Evaluation Explorer Sensorgram b r All sensorgrams Plot bee lid Baseline Sample Bee a Binding level Binding stability g Binding to reference g Darry over gg Controls binding g Controls stability 7 Solvent Correction A Solvent Correction Biacore T100 5 227 5 79
149. 2 boseline_2 binding 2 X stability 2 RelResp baseline_2 RU Toolbar Reference line Menu bar View Reference Line amp 7 l Biacore T100 2 21 Te Biacore T100 Control Software manual blr ilz Fie Edit View Commands Run Tools Help x mm p S E Cyde 1 Curve Sensorgram Fc 1 axlo CEE C Lock scale cj M 258 0 s 58051 5 RU x binding 1 amp 45000 Wash Starf stability 1 Inject Read Wash Ready co e e 50 100 350 400 AbsFiesp SD LASD Slope elesn Baseline Id Keywords in cycle 1 Value 5 3583950 002 2003 0 00 0 0 baseline_1 5 580110 2 00 014 1 06 22115 0 binding 1 5 358852 054 0 60 004 0 8 stability_1 Flow 30 Flow Path 1 Online COM1 Temperature 25 00 9C Running manual run Sample compartment temperature current 25 C set 25 C Run time 7 min RE
150. 93 5 A d 94 5 2 2 kk 109 CEA Dor nc eO cT 123 Bess UD CT OAE NEEE eee ated Sedet 124 SC M siamo N aaa E T N 134 5 4 ee 143 CR IMP E N E X a AOE EEE EAEE 148 DAT O N annn T O a TAE 165 5 5 kk 174 lS ik 174 5 5 2 kk 183 5 6 193 5 6 1 B AD ANODE S a 195 D MOM MIC S AE E E AAE GG 203 5 7 211 al co USE T saciseteerisseeriietteterteriocl beta tede beet NE E AE 214 2 224 5 8 1 0 a a na D E 233 ED DR TTD ERR 234 6 247 6 1 Lk 248 02 D EU oaa E E S T 249 ELT QUE SY MSS O T NOE TENERA ERR 270 Biacore T100 PES Deae ue 214 7 1 kk 277 Te CO Nm aii oO 277 7 1 Z Desorb and Sanitiz coute Fever ba etre ubere roce epe bec ch edet abe a ie iie eee be b dee lo Red 278 CSUEMOIV BURST TUDIN senectt due dte o rl uen aun cfc d un n sve e UR ct ui urs 281 EA WIERE BUN SETODINO sei E 283 7 2 285 sp ed NGFETIGI
151. F Allsen 3 Window gt Tile Horizontally Tile Vertically Evaluation Explorer Biacore T100 5 83 5 15 Multiple Rmax 1 ES Kinetics Affinity Select Curves Create Select evaluation mode Single mode Batch mode Curves Curve v Ligand Sample 89 w Temperature low Contact Time Diss Time l min s s 600 0 600 1 600 0 600 0 600 0 600 1 600 0 600 0 600 0 600 1 600 0 600 1 600 0 600 0 Zoom lock Show concentration series Show blank s AOU F Kinetics 7 Affinity Select Curves Create Select evaluation mode Single mode Batch mode
152. 107 Save Results From Run s Save in e CSK E immobilization of antibody blr v My Recent Documents 3 Desktop My Documents qi My Computer File name s CK antibdy antigerl MyNewok _ Save as type Pesut fle bi Save in File name Sqve T Biacore T100 Control Software SCK_antibdy antigen blr BEES iz File Edit view Run Tools Help x i eb El 20 I0 RE evde 6 Curve Subtracted Fc 4 3 RU C Lock scale 546 544 542 0 200 400 600 1200 Time Window AbsResp SD LHSD Slope RelResp Baseline Id i Keywords in cycle 6 Value 51 8 176 8 131 8 364 7 379 7 552 8 567 8 740 7 755 7 5 015 015 003 baseline AssayStep Sample 5324 0 11 D 11 0 02 binding AssayStepPurpose Sample 531 4 0 22 0 22 0 02 stability Buffer Buffer 535 0 0 14 0 13 002 binding_2 CycleType Sample 534 1 0 17 017 0 01 stability 2 Sample 1 Conc amp 1 1 063 5378 015 015 6003 binding 3 Sample 1 Conc H2 2 125 537 0 0 15 015 0 01 stability 3 Sample 1 Conc H3 425 5405 0 15 015 0 01 binding 4 Sample 1 Conc H4 8 5 5334 0 15 015 0 03 stability 4 Sample 1 Conc H5 17 328 8 5434 0414 013 0102 binding 5 Sample 1 Ligand antibody 343 8 5420 0 12 0 12 0 01 stabilitu 5 Sample 1 MW
153. 5 137 Calibration Curve deve Em OD Su iate 2 Cure 1 kal Ctrl Calibration T able Calibration Curve Conc Response Calc Conc ng ml RU ng ml RU Biotin 1380 5 0 9948 1600 1380 5 0 9948 1400 2 1298 6 1200 1000 800 Relative Response 600 400 Parameters 200 lt Dverlay gt 20 20 40 B 8 100 120 Concentration nami Calibration Table Conc Calc Conc CV CV 90
154. 5 155 T Method Builder Main Define variable handling for each amp ssay Step General Settings Sample CO Define all values at run time Assay Steps O Define all values in method Define some values in method and others at run time Cycle Types Variable Settings 7 Values for these variables are defined in the method Values for these variables will be defined at run time Command Variable Command Variable d L Sample 1 Blank i Sample solution Flow rate min MW D 209C SeupBun Dilution Enter values for variablx defined in the method These values will be used for all cycles in the assay step Assay Steps Sample amp O Define some values in Method and others at run time 5 Setup Run Variables Sample solution Flow rate ul min 5 ul min 100 ul min 2 MW Do 35 D 20 C 20C Dilution d zh cp Blank
155. Ctrl Breakl _ Biacore T100 Evaluation Software Biacore T100 134 5 5 3 2 Evaluation Biacore T100 Eyaluation Software Concentration Analysis blr AJ Kinetics Affinity Concentration Analysis gt Thermodynamics s Assay Step Purpose verlay e Cycle Overlay ensorgram aJ All sensorgrams RU Sensorgram nz E Plot 44000 oom Lock Baseline Sample Binding stability Controls stability Report Point T able mm Report Point Table 43000 43500 42500 42000 Calibration 41500 Control Sample Sample p 41000 Startup 40500 40000 39500 39000 0 5 34 Keyword table Tools Keyword Table S Keyword Table 2 E
156. Start Save as Methods and Templates 2 Bia Users Don t Save Save in File nme Save Standby flow Biacore T100 Evaluation Software HB Biacore T100 272 6 6 6 Control Sample R1A1 735 R1A12 12
157. The method has been verified and can be used to setup a run Variable Settings Cycle Types gt The Method has been verified and can be used to set up arun Setup Run T Method Builder Detection Detection Flow path v Flow path Next gt Biacore T100 158 5 Fe Method Builder Variahles Variable values for Assay Step Sample EE mt 00000 Sample oeon How rate mi MW Da pG Diution mouse IgG x1 6400 150000 5 09E m 2 mouse IgG x1 6400 1 150000 5 09E 11 3 mouse IgG 1 1500 150000 5 09E 11 4 mouse IgG x1 1600 150000 5 09E 11 6 mouse IgG 1 400 150000 5 09E 11 7 mouse IgG C 100 150000 5 09E 11 8 mouse IgG 1 100 150000 5 09E 11 mouse IgG x1 400 150000 5 09E 11 sample 2Uv2d 2 Sample solution Flow rate Ml min 5 ul min 100 ul min 2 Mw Da 7 3 58 D 20 C 20C Dilution l
158. 3 D SingleDualMulti Singe L 2 5 4 Dual Ls 3 4 ls 4 5 Multi 12 544 2 1 4 3 2 1 5 1 4 1 Sample compartment temperature 4 45 C 25C Concentration unit 5 Buffer settings After run Assay Steps Biacore T100 96 5 T Method Builder Main Startup General Settings Startup Startup 3 times as entered m AssaySteps i a E Copy Sample 5ample Sample 1 time as entered Cycle Types Variable Settings a Move Up Move Down Cycle Run List Assay step properties Base settings Recurrence Name C Repeat assay step within Purpose Startup v Every d Startup w Distribute occurrences evenly Hun assay step once first Run assay step once last cycle Assay step preparations 5er of replicates Temperature 3 times Buffer As entered 1 2 3 1 2 3 Order 1 1 2 2 3 3 Random Hep sw jJ Sse amp Startup Number of repli
159. Biacore T100 5 131 5 32 Control Sample R1A1 R1A12 12 T 060327 Thermo Rack Positions Reagent Rack 2 Type Sample 1 1 148 Negative control Control sample 40 i 148 Negative control Control sample 148 Negative control Control sample i 148 Negative control Control sample i 148 Negative control Control sample E j OGO 148 Negative control Control sample 1 Oe 148 Positive control Control sample A F G i 148 Positive control Control sample 96 Deep Well Microplate 148 Positive control Control sample 148 Positive control Control sample 148 Positive control Control sample E e Q L Q O O Q O 148 Positive control Control sample T e Q O O C 148 Analyte A Sample 10 i 148 Analyte A Sample n OQ e C C C C C C i 148 Analyte A Sample o Q s Q Q s 148 Analyte A Sample 9 Q Q Q Q O O Q OQ 148 Analyte A Sample 7 O Q Q O O O O O 148
160. ka 1 Ms kd 1 s KD M Rmax RU Conc M tc 1 531E 6 0 002610 1 705E 9 7 178E 7 1 100E 9 2 231E4 8 2 200E 9 2 231bE 8 X 0 2011 4 300E 9 2 231E4 8 0 1965 8 500E 9 2 231E4 8 0 1167 1 700E 8 2 231E 8 0 1062 Finish Add Fit Model Fit LA Kinetics Affinity Fit Kinetics Create Curve Fc 2 1 Add Fit Model Two State Reaction css Report Residuals Parameters kal 1 Ms kd1 1 s ka2 1 s kd2 1 s KD M Rmax RU Conc M tc Flow ul min kt RU Ms 1 554E 6 0 002932 5 060E 4 1 866E 6 5 790E 12 7 145E4 7 30 00 2 2ZUE 8 30 00 2 2ZUE 8 30 00 30 00 30 00 B amp ATULISd Ncof sid REELT Current Fits Current Fits Finish Biacore T100 F Kinetics Affinity Fit Kinetics Create 5 79
161. 5 7 Menubor Toolbar Evaluation Explorer Work area Menubar Toolbar Evaluation Explorer W nsorgram i plot Baseline Binding level m Report Point T able Binding stability Binding to reference Work area 5 67 AbsResp Baseline 73 50 RelResp Bqseline RelResp Binding level Evaluation Explorer Biacore T100 68 5 Toolbar qp Kinetics Affinity 4 51 y pg A Surface bound Kinetics Affinity Select Curves Create Select evaluation mode Single mode C3 Batch mode Curves Curve Ligand ribody 10ug7nli v Sampie a Cycles Conc Flow Contact Time Diss Time AME nM ulmin s s 1 1 30 za 3n 5 L4 v v v L4 L4 v L4 Shown concentration series Show blank s Show average blank s
162. Biacore T100 60 5 Te Kinetics Affinity Samples Samples Sample id a 080 HH 0 01265 0 02530 0 04945 0 08775 0 1955 0 01265 HH 000i 0 01265 a 02830 a 04945 0 08775 0 1955 0 01265 a OO HH Run order 9 Azentered O Increasing concentration Sample id MW Do Concentration nM ug ml NH Next gt 5 0 0 7 1 2 Next gt Recommended settings are nat followed Sample serie sample The sample series should contain at least anie sample with zero D concentration The sample series should consist of at least five 5 different concentratans The sample series should contain at least one non zero concentration that is to be run at least two 2 times
163. 2 Menu bar Commands Eject Rack Eject Rack Tray Hack Trav Ejected Click DK to retur the rack tray to the sample compartment Time tn auto close 50 OK Inject Vial wel position R2 B1 8 amp Beastin Minimum required volume in vialwell For this injectien 58 ul Inject command OK T Biacore T100 Control Software manual blr ilz File Edit View Commands Run Tools Help id ES Bl E e Cycle 1 m Curve Sensorgram Fc 1 I ZIZI B C Lock scale E9 Inject R281 60 baseline 1 50 100 250 Time Window amp bsResp SD LASD Slope HelHesp Baseline ld Keywords in cycle 1 Value 210 0 5 358350 002 003 0 00 0 0 baseline 1 275 0 5 580110 2 00 014 1 06 22115 0 binding 1 290 0 5 358352 0 54 0 60 004 0 8 stability 1 Flow 30 Flow Path 1 Online COM1 Temperature 25 00 9C Running manual run Sample compartment temperature current 25 C set 25 C Run time 6 min
164. Flow path Biacore T100 268 6 LS Method Builder Variahles Assay steps Startup Sample Control sample Variable values Far Assay Step Sample RR ETT TE sisi ed S EB n 1 95 7 81 31 25 n 125 5 2000 bal Sample amp Assay Setup Define all values at run time 6 4 Excel 2 Excel Excel txt Next gt l Biacore T100 6
165. RelResp Binding level Evaluation Explorer 5 185 Binding level 2 Evaluation Explorer Plot Binding level CuveNamere 43 J Assay Step Purpose z verlays h Cycle number lt 0 werap h Ru Binding level F ZoomLook MoE EE EN 17 1 12 5 8 2 094 4 8 20 0 3 0 4 3 6 7 4 zx c Control Sample Sample Startup Relative respos binding 3n 40 Cycle number ZU F Esai Nams re ua e20v2L value Yalue
166. B 2 Heterogeneous Analyte A1 B A1B A2 4 B A2B 1 2 Heterogeneous Ligand A B1 AB1 A B2 AB2 2 Two state Reaction A B AB AB 1 Kinetics Affinity Fit Kinetics Create Curve Fc 2 Ligand antibody 10ug ml pH Sample antigen Temperature 25 LC Add Fit Current Fits Model 1 1 Binding v 1 1 Bindini Mi add Quality Control Report Residuals Parameters Biacore T100 5 73 1 1 binding Quality Control z 5 10 5 10 Quality Control 5
167. Biacore T100 AN This will stop the run stop Run Run Stopped Finishing current cycle please wait Abort cycle bu Cti Break Ctr Break Biacore T100 Evaluation Software Biacore T100 5 223 5 75 binding stability
168. IS fi LL C Recurrence Number of replicates Biacore T100 258 6 Cycle types Te Method Builder Main Description of selected cycle type Solvent correction New This cycle is used in startup sample and control sample steps General Settings LMW kinetics Contains injection of sample and carry over control running buffer XK Delete Odelpe gt Variable Settings Verification Commands Report Points Capture Settings for Sample 1 Type High performance v S Method Variables Evaluation Variables Set property as variable Sample solution Is variable Sample 1 Solution SeupHm rm control 1 Contact time eo s C Contact time s T GOO Ma C Dissociation time s C Flow rate pl min Flow rate 30 pl min Estra wash solution Flow path Predip d Mix with Stabilization period a Estra wash after injection with 50 22 DMSO C Stabilization period D s Cycle types 4
169. Multiple Rmax 5 15 Biacore T100 92 5 5 18 Quality Assessment Ko 1 2 CREAR C Rmo D x kKp M RU B 5 4 3 B 20 1 1g E z B B 3e B 4e B 5se B be B Te oe Je 1e 5 Concentration Ii Report Parameters Lb 9 Rman RU offset Ru Ch us a b41E 7 0 0545 2 05 0 02560 AFOJ DCREDRIROBSCIA ESSET Z2SBU C PIE RES kR SED 4e 5e 6 Concentration Report Parameters Rmax RU offset RU Chi RU KD M Rmax RU offset RU Chiz RU2 o 701E 0 524 943 Biacore T100 5 93 5 2
170. 6 Biacore T100 224 5 5 7 2 Evaluotion 5 1 nb Sensorgram Cusen Eger d F All sensorgrams m A dit PANC ane Curve Name Fc 4 3 JP Assay Step Purpose Overlay Cycle Overlay J F Sensorgram j ll sensorgrams RU Sensorgram E Plot 8800 O Zoom Lock Baseline Sample Binding level Binding stability Binding to reference Controls binding 8400 Controls stability gt Report Point Table mj Report Point Table 8500 8200 5000 Conditioning L d L 3 Control Sample 7800 Sample T Solvent correction 7600 Startup 7400 7200 4 7000 6800 Time 5 77 Keyword table
171. Low Sample consumption 7mm 25 ule High performance 7mm 58 ul Single cycle kinetics 5 7mm 58 ul ls variable s fratge S umin Detection Dual First 2 1 1 4 3 3 Second 2 1 2 4 3 4 Biacore T100 260
172. pH Biacore T100 212 5 SPR R UU 100 RU
173. 1 2 Carry Over Next gt Te Kinetics Affinity Setup Conditioning Bun conditioning cycle Contact time Ea s Mumber of injections Startup Bun startup cycles Solutio buffer Humber of cycles Solvent correction Hun solvent correction Number of injections Repeat after 20 sample cycles Conditioning Solution contact time s Number of injections Biacore T100 216 5 Startup Solution Number of cycles 3 Solvent correction Number of injections Repeat after Sample cycles Next gt Te Kinetics Affinity Injection Parameters Sample Contact time s Flow rate am
174. BlAdesorb Solution 2 20 ml Start l 2 3 Wash Buffer Tubing Step 3 Wipe the tube with a moist tissue Place buffer ar water on the left hand tray and insert the tube sn 141 2 i iuWXKNJUHIEATL Start l 3 Wash Buffer Tubing The wash Buffer Tubing procedure is completed Lose Close Biacore T100 7 285 7 2 7 2 1 Normalize Biacore Maintenance Kit type 2 BlAnormalizing solution Menu bar Tools More Tools Maintenance Tools Normalize Stqrt i Harmalize This procedure normalizes the detectar signal Total run time is about 8 minutes Req
175. Excel 2 Excel txt c Te Thermodynamics System Preparations Frime before run Normalize detector Es m Prime Normalize l T Thermodynamics Rack Positions Reagent Rack 2 Type Sample 1 Sample 1 Conc nM MW Da 118 antigen Sample 11800 e e 118 antigen Sample 11800 118 antigen Sample A 11800 118 antigen Sample 11800 118 antigen Sample 11800 118 antigen Sample l 11800 1 118 antigen Sample 11800 ee HM f 118 antigen Sample 11800 AN m D E F 6 118 antigen Sample 11800 96 Well Microplate i 118 antigen Sample 11800 118 antigen Sample 11800 118 antigen Sample 11800 E Q Q Q OQ Q OQ Q Q 118 antigen Sample 11800 1 x X XX 118 antigen Sample 11800 60000000 I Q Q OQ 118 antigen Sample 11800 O00QO 60o00 118 antigen Sample 11800 118 antigen Sample 11800 6 2 e t x 118 antigen Sample 11800 i 118 antigen Sample 11800 3 O OO Q C O C 118 antigen Sample 8 5 11800 4 e Q O e e C C C 118 antigen Sample 17 11800 118 ant
176. TEL 03 5331 9336 AL ERIT 9 00 17 30 IB sis ste seBUC FAQ Q amp A Web pFAO Q amp A Web O0 TEL 03 5331 9336 9 00 17 30 0 O W FAX 03 5331 9370 e mail Tech JP ge com Life Sciences Academy gt lifesciences ac com Bicss779 90 EA TEL 05 5551 9515 FAX 05 5551 9549 BAR tEeusu TEL 03 5331 9330 FAX 03 5331 9324
177. BIAdesorb solution 1 4 BIAdesorb solution 1 Dd mA CE ODIO E v ad 4 CHRIS Biacore T100 276 7 1 Toolbar Eject Menu bar Tools Eject Chip Hiacnre T100 AN This will eject Ehe sensor chip perde Eject Chip Sensor Chip Maintenance Insert Chip 9 Mew chip Q Reuse chip Mew chip Chipid OBDEO9 0135 1387521 Beck crie Insert Chip Chip type Maintenance Chip id L Dock Chip Dock Standby flow Dock Prime Biacore T100 7 277 7 1 7 1 1 Desorb IFC
178. Constant Initial value 0 Do Parameter Setting OK Fit Biacore T100 80 5 TAE 5 13 Current Fits Current Fits 1 1 1 Binding Description Two State Reaction Delete Biacore 100 Evaluation k J Are you sure vou want to delete this Fit OK Curent Fits 1 Two State Reaction Description Biacore T100 5 81 5 14 Batch mode Select Evaluation mode C Batch mode Batch mode F Kinetics Affinity Select Curwoe Create Select evaluation mag
179. L urve Name Fc 2 1 h lt Assay Step Purpose D verlays Cycle lt 0wverlay gt h 4 Curve Name Fe 2 1 J Jo i UL eal a2gs2L Bto20 tv Reference Active Referencesubtracted Ctrl F All semsorgrams PE Curve Names Overlay rj 4 Assay Step Purpose verlay v Cycle Overlay rj RU Sensorgram 38000 Zoom Lock 37500 37000 36500 36000 Sample Startu p 35500 35000 34500 34000 Biacore T100 296 9 6 Cycle Overlay 7 9 Jo 35 U eral Ju 51 y 2L Do er Lyclez Assay Step Purpose Sample mcm 1 Startup Buffer hausmsaeusunusuauuuumsuuuuuansu RARESERI SAEERERISAREREBARI
180. Le Menu bar Commands Inject Inject Vial well position A1 A1 Peas u Minimum required volume in vial well for this injectio Vial well position contact time Inject T Biacore T100 Control Software manual blr PUE iE Fie Edit View Commands Run Tools Help B8x ri TT 4 E N Cycle 1 g Curve Sensorgram Fc 1 FE iI8 i9gIBl RU C Lock scale 35840 amp 35900 Inject Vial well position R141 5 Eentact time CR M M j9 0 0 Minimum required v pil 0 10 20 8 90 1010101010101010 Fc Time Window AbsResp SD LASO Keywords in cycle 1 Value Flow 30 Flow Path 1 Online COM1 Temperature 25 00 9C Running manual run Sample compartment temperature current 25 C set 25 C Run time 2 min Cancel Inject Biacore T100 2 19 Eject rack tray
181. Ligand 98 ul 7 mm Eject Rack Rack tray port OK Eject Rack Tray Rack Positions Next gt l Te Immobilization Prepare Run Protocol Prepare Run Protocol Make sure the correct sensor chip is docked Make sure all samples amp reagents are loaded in the rack and microplate according tn the Rack Positions setup iVials should be sealed with rubber caps and microplate with adhesive foil Place the buffers on the left hand tray and insert the correct tubing s see below Mote Standby after run will use buffer A Make sure there is fresh water in the water bottle nh the right hand tray If necessary empty the waste bottle before start af the run Estimated run time 31 min excluding conditional statements temperature changes and standby How Estimated buffer consumption a Running buffer jj Approximately 100 ml Start
182. RI RI Biacore T100 74 5 Residuals Y Residuals for a poor fit RU Residuals for a good fit RU Biacore T100 5 75 Report Kinetics Affinity Fit Kinetics Create Lurve Fc 2 Ligand antib
183. Series S Sensor Chip NTA 3 BR 1005 32 Biacore T100 1 9 1 2 2 Menu bar Tools Prime Tools Help Prime Shutdown Start Place buffer on the left hand tray and insert tube amp Place water on the right hand tray and insert the water inlet tube Prime Prime Friming please wait Time left QD OB 46 Prime The Prime procedure is completed Close Standby flow 1 8 Prime Biacore T100 10 1 1 2 3 Analysis temperature Menu bar Tools Set Temperature Tnnl Biaco
184. Biacore T100 7 281 7 1 3 Empty Buffer Tubing BC D Buffer scouting BC D 20 70 Maintenance Tools Empty Buffer Tubing Start amp 2 v 2d 2 Empty Buffer Tubing This procedure empties all Four buffer selector inlet tubes The procedure is divided inta three steps Total run time is about 20 minutes Required salutians eionized water TUZ ethanol Next gt Empty Buffer Tubing Step 1 Place a bottle containing deionized water on the left hand plate and insert the four buffer inlet tubes ABCD Start 1 2 Empty Buffer Tubing Step 2 Place a bottle containing at least 10 ml 73 ethanol an the left hand plate and insert the Four buffer inlet tubes stat
185. Empty i Mix min capacity 1144 Control sample Mix ALLALI L IO CO Q Empty Mix min capacity 114pul Control sample Mix ececc0000 113 501 2436 01 Sample e amp e amp e C 113 501 2432 01 Sample 113 505 n001 03 Sample e e e e e 113 PET n1 Sample v 3 Heb Mew v Bak Neo Cos D 5 31 Empty 5 Mix min capacity 1144 Control sample Mix Empty i Mix min capacity 1144 Control sample Mix Menu Export Positions Menu Simple Position Import
186. RU Sensorgram Zoom Lock n1 1230 5 e m5 a c 1225 Z PE Cc 2 1220 c amp 1215 s m5 n d i T n pi g TAN i m be 1200 50 100 150 200 250 id Biacore T100 298 9 9 3 3 Sensorgram window _Tcck Tools gt Sensorgram Adjustment 7 v 2d amp E Adjust Sensorgram AX Adjustment Of NA CO Report Point time D CO Injection E vent me Y Adjustment Report Point response Q Injection Event response 0 Blank Subtraction A Enable Blank Subtraction X Adjustment v Report point time 0 baseline AOAdpustment CQ or A Repo Point ime 0 baseline atabili OK RU Adjusted sensorgram 1230 i bindir 5X stability 1225 binding stability ity 1220 w Drfdtimpling binding binding z stabilite stability Fi binding stability 50 50 100 150 200 250 Time 0 baseline s Biacore T100 9 299
187. Repeat assay step within s j NNI Assay step 1 Assay step 1 Base settings Base settings MH ame Purpasze Connect In cycle type Name Purpose Connect to cycle type Solvent correctior Solvent carrection amp olent carrectian Solvent correction Solvent correction Solvent correction Recurrence Number of replicates Assay step preparations Biacore T100 266 6 Variable Settings Te Method Builder Main Assay steps Define variable handling for each amp ssay Step General Settings Sample O Define all values at run time Solvent correction Astay Steps Control sample Define all values in method Cycle Types i Define some values in method and others at run time CceTvpes Variable Settings 2 Enter values for the variables in this assay step Verification
188. 2 1 1 Kinetics Mw km Bivqlent Analyte CFCA _ Christensen Anal Biochemistry 1997 249 p 153 Sigmundsson K et Al Biochemistry 2002 41 p 8263 Biacore T100 144 5 CFCA 738 Z 5000Da 2 Ko 107 gt ka gt 5X104M is1 3388 150kDa TIA 5 000RU CFCA 0 05 5 ug ml 1 ug ml 280 nm 10
189. Test Tools gt System Check Start System Check Select tests to run This procedure should be run at 25 C with a new Sensor Chip CM5 and with HES as running buffer Choose Close if you need to change the sensor chip reset the temperature or change running butter A Reagent pumps and blank injection B Hafractameter performance and Fow cell leakage C Injections D Noise E Mix F Buffer selector A Tests if the peristaltic pump is in order and that a sample injection with buffer from the Reagent supply black is all right System Check Next gt Buffer scouting F Biacore T100 7 287 System Check IF the buffer selector shall be tested test F put tube B C and D in water IF all inlet tubes are nat intended tn be used after System Check don t forget to runi Empty Buffer Tubing after the test A HBS N Buffer 10x BC D Buffer Scouting BCD
190. 148 Positive control Control sample i 148 Positive control Control sample iE Q e Q Q Q Q O O 148 Positive control Control sample 00000000 448 mateA Sample 10 Q e O O O O O Q 148 Analyte A Sample 148 Negative control Control sample Oo 0 0 0 0 0 0 Cn o e 148 Analyte A Sample Fu e 148 Analyte A Sample 148 Analyte A Sample 148 Analyte A Sample 148 Analyte A Sample 148 Analyte A Sample 148 Analyte A Sample 148 Analyte A Sample 148 Analyte A Sample 148 Analyte A Sample 148 Analyte A Sample 148 akaka A Sample ce b aio Menu 7j 5 Automatic Positioning E25 Eo Te Automatic Positioning Change the order in which the samples are positioned by ordering the regions The first region in the list is positioned first Region Color Orientation Anchor Rack Vial Sife Pooling First Sort By Control sample E Cyan Column Bottom left Sample Small i Ascending Sample E CarkElue Column Bottom left Sample Small Ascending Startup BEI crimson Column Bottom left Sample Small Ascending Wash Yelaw Column Bottom left Reagent Large Ascending E Small Content Ascending Y Solvent correction buffer ll Blue Column Bottom left Reagent gt Pooling Auto
191. Biacore T100 5 161 5 43 Control Sample R1A1 R1A12 12 Te 060327 Thermo Rack Positions Reagent Rack 2 Type Sample 1 1 148 Negative control Control sample 40 i 148 Negative control Control sample 148 Negative control Control sample i 148 Negative control Control sample i 148 Negative control Control sample E j OGO 148 Negative control Control sample 1 Oe 148 Positive control Control sample A F G i 148 Positive control Control sample 96 Deep Well Microplate 148 Positive control Control sample 148 Positive control Control sample 148 Positive control Control sample E e Q L Q O O Q O 148 Positive control Control sample T e Q O O C 148 Analyte A Sample 10 i 148 Analyte A Sample n OQ e C C C C C C i 148 Analyte A Sample o Q s Q Q s 148 Analyte A Sample 9 Q Q Q Q O O Q OQ 148 Analyte A Sample 7 O Q Q O O O O O 148 Analyte A
192. Include Include OK 5 47 CFCA Initiqal rate RU s 0 3 15 RU s lt 0 3 RU s gt 15 RU s 2 QCratio z 0 13 lt 0 13 Biacore T100 5 169 ES Calibration free concentration Select Samples Create Expand all cycles Uze reference subtracted data Fr 4 3 1 Dug ml Protein pHE O0 mause I
193. Evaluation Explorer gt Work area Menubar Toolbar Evaluation Explorer W nsorgrom piot Baseline Binding level Binding stability Binding to reference Work area Biacore T100 m Report Point T able Sensorgram window AbsResp Baseline 73 50 RelResp Bqseline RelResp Binding level Evaluation Explorer 5 167 s Concentration Analysis 7 A Calibration free l2 Toolbar Calibration free concentration Select Samples Create Expand all cucles Uze reference subtracted data Fc24 3 100ugiml Protein A pH5 0 mouse IgG x1 6400 z5 150000 Fc 4 3 100ugiml Protein A pH5 0 mouse IgG x1 1600 1600 25 150000 Fc24 3 100ugiml Protein A pH5 0 mouse IgG x1 400 z5 150000 Time
194. Next gt To Method Builder System Preparations Frime before run Normalize detector Es Es Prime Normalize Temperature settings Analysis temperature 25 C Sample compartment temperature ZEE Next gt Biacore T100 242 5 T Method Builder Rack Positions Empty Recovered sample min capacity 20pl 819 0 5 TFA 521 0 595 TF 427 50mM NH4HCO3 eicere O00 C O0 O0Q0Q0X OOO 209990 OOO O OO 000000 lt Back New gt gee ul Biacore T100 5 243 5 83 Control Sample R1A1 735 R1A12 12
195. Open Browse Open Te Kinetics Affinity Injection Sequence Detection Chip Flow path Chip type Capture Regeneration Cary Over 1 Biacore T100 5 215 Detection Flow path 2 1 4 3 Chip Chip type Capture 2 4 5 76 Sample P424 kom Regeneration
196. z eport Point T able ri epo Point T able A Solvent Correction binding binding binding binding binding binding binding binding binding binding binding binding binding binding binding binding binding binding binding binding binding binding binding binding binding binding binding binding binding 8206 9 8174 0 8145 2 8118 2 8093 7 8031 0 7999 7 7916 2 7939 0 7943 9 7950 3 7950 2 7940 0 7924 5 7905 8 7885 2 7743 3 7671 5 7611 4 7591 1 7571 6 7552 8 7534 6 7515 2 7496 3 7473 8 7463 7 7446 5 7428 7 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 0 04043 0 0974 0 06585 0 009818 0 02562 0 006455 0 05382 0 03872 0 0676 0 1028 0 06665 0 03736 0 03608 0 0303 0 008681 0 0315 0 1533 0 02218 0 01423 0 006287 0 01462 0 01823 0 01102 0 01717 0 01661 0 03216 0 002334 0 1079 0 009381 Fc Report Point binding Assay Step sample FIResp RU 2 Biacore T100
197. Evaluation Explorer 5 68 Evaluation Explorer i Kinet Backspace i73 Kinetics Affinity pun TEIG Biacore T100 208 5 Toolbar A Kinetics Affinity ES Kinetics Affinity Select Curves Create Select evaluation made Single made C3 Batch mode Curves q Contact Time Diss Time s s 1 06 30 2 13 30 4 25 30 30 30 30 30 30 w La La L L L4 La La La Show concentration series Shaw blank s Show average blank z Multiple Amas Adjust Inje
198. Heb Bak Meo ce Conditioning Solution F3 E Te E RR BT CD 08 HN contact time s Number of injections Biacore T100 176 5 Startup Solution Number of cycles 3 Next gt 5 51 Sample 4 Ta Binding Analysis Injection sequence Capture SAMPLE 1 Sample EH SAMPLE 2 Enhancement SAMPLE 3 Iv Regeneration REGENERATION 1 Help 3 2 Biacore T100 5 177 Te Binding Analysis Injection Parameters Sample Contact time s Elow rate l min Dissociation time s Regeneration
199. Running buffer J j Approximately 200 ml EUCKRBVGOERESIR AMERA 59BERLLILO B ABIIT Start Save as Methods and Templates 7 Bia Users Don t Save Save Results From Run s Save in E T100manual oO Din 4 Immobilization of PrteinA blr My Recent manual blr Documents pHscouting blr regeneration check blr regeneration check wizard blr Desktop surface parformance wizard blr My Documents da My Computer File name Thermodynamics antibody vs antigen bc a J My Network Save as type Result file blr v Biacore T100 202 5 Save in File nime Sqve Temperature Adjustment Waiting for stable temperature To obtain reliable results vau should wait until the temperature is stable Set temperature a Current temperature 23 09 Temperature stable Unstable
200. Solution 1 rmid Gl H IpH2 High viscosity solution Contact time s Flow rate julmin Stabilization period o s Sample contact time 5 Flow rate Umin Dissociation time RBS s Regeneration Solution High viscosity solution 40 contact time s Flow rate ul min Stabilization period s Next gt te Binding nalysis Samples Sample table Sample id 1 D I Ch CH A i aG e mm Sample id Control Samples Biacore T100 178 5 Te Binding Analysis Control Samples Control sample definition Run control samples Repeat control zample s every sample cycles Control samples Control sample id 1 Control Repeat Control Samplels every OK Next g
201. X WWwW gelitesciences co p TT wx A 2 2014 GE GEN VAT EAD 77 T169 0073 me Tu 3 25 1
202. nd Templates Name 070814 3070813 3080314 30805 om nw un t tv EEO sS t50ctco IPE g mo orog E oOoo0S50 5 xtUuu EE e o 0830 T O0 mz kA 9 GS PEZ T Tes maf EO 20 zm 50 2 amp 8 oou ESES v Sos5mss5z e o m 3 m D 2 e S UE o o D 2 2 A 292 z 2 2 2 5 1 A J Con A v Ass A A A M Assay Thermodynamics New Methods and Templates 2 Open Browse Open Te Thermodynamics Injection Sequence Detection Chip Elow path Chip type Capture REGENERATION 1 Regeneration Carp Over 1 Biacore T100 196 5 Detection Flow path
203. ul l Eject Rack Rack tray port Eject Rack Tray Hack Trav Ejected Click DK to retur the rack tray to the sample compartment Time In auta clase 50 OK Eject Rack Tray Rack Positions Next gt Biacore T100 3 31 Te Immobilization pH Scouting Prepare Run Protocol Prepare Run Protocol Make sure the correct sensor chip is docked Make sure all samples amp reagents are loaded in the rack and microplate according tn the Rack Positions setup Mials should be sealed with rubber caps and microplate with adhesive foil Place the bufferis an the left hand tray and insert the correct tubingis see below Mote Standby after run will use buffer A Make sure there is fresh water in the water bottle on the right hand tray If necessary empty the waste bottle before start of the run Estimated run time 18 min excluding conditional statements temperature changes and standby Flow Estimated buffer consumption ad Running buffer j j 1 4 Approximately 100 ml EUCRSVGIERASSIB AMEA 5 3SEERb u 2f
204. 190 5 E 5 59 Evaluation Explorer Plot controls binding mU Contras birding fnm Lact m vun Eod e s Datt B cikg rema m ras Dinar Evaluation Explorer Plot Binding to reference Dji Birding ra reference 1 Zur bx u a u mh amp Xr v Cobi map p Biacore T100 5 191 5 60 Tools gt Custom Report Points S Custom Report Points Custom Report Points Position Add S Add Custom Report Point Report point ld stability Window 5 v s Position the report point 20 seconds after v Sample 1 injection stop Calculate response relative to report point Cycles Apply To Selected Assay step purpose Selected Assay step purpose Startup CO Apply To Selected Cycles Calibration
205. 4 SCR 5 ul min 100 ul min CFCA IgG CFCA gG IgM CFCA CFCA 5 40 mol mzs x km
206. 54 55 56 75 91 92 93 117 193 194 205 211 Kev TODE T PTU TPTO O TT 66 87 109 134 165 183 203 224 NEETI LR ANE 85 aie fien MT P PE 53 57 214 262 i 143 M Aj e IR IBI RESI mS 14 15 47 Method M 22 55 42 94 148 234 248 261 262 Wi ceres Toldos e RE EH GG 261 262 Biacore T100 AA SAU MGIe SG 248 Methods and Templates 28 31 34 38 41 57 64 106 124 132 162 174 181 195 201 214 221 244 248 271 Mi eU erm 126 260 Piu qun EM M MM LIE 0 at 85 91 N QI deo M CC P 5 Mie m M 23 25 26 33 35 36 38 40 44 Wielgnqreijrz sor a 29 37 61 103 129 160 178 199 218 241 270 285 302 Number Orey CE S ainia 59 126 NUmMDEr Or s edd e IER EA EERIE O MUT 59 125 197 216 Number or lreplICaleSnn iit ata e LA EB EAR 96 97 150 151 152 237 238 254 257 265 O OO REMO Dao Ni 86 Qui TTE 94 148 234 249 P mau ER 9 29 37 61 103 129 160 178 199 218 241 270 276 277 280 286 289 RS 22 PU OS Ri 253 254 265 Q OUO ASSESSED E EM MM M MM MM MM MM M MM 92 Quali X OM O i 73 116 R ROCK TTO ii ii 12 13 30 38 64 106 132 162 181 201 221 244 271 FORLAG CC T T 1
207. 88 sample 1 Sample 88 sample 1 Sample 88 sample 1 Sample 88 sample 1 Sample 88 sample 1 Sample 88 sample 1 Sample 88 sample 1 Sample O 000000000 88 sample 1 Sample 88 sample 1 Sample 88 sample 2 Sample 88 sample 2 Sample 88 sample 2 Sample 88 sample 2 Sample 88 sample 2 Sample 88 sample 2 Sample w A Cn n 09000000 QOO 88 sample 2 Sample 88 sample 2 Sample 88 sample 2 Sample 88 sample 2 Sample DOOOOOOOOO000 gt ge C Cx Le J oe D 6 5 Menu Export Positions Menu Simple Position Import
208. Curves Curve Fc 4 1 v Ligand Sample Temperature Flow ul min Zoom lock Show concentration series Show blank s C Show average blank s Curve Add Rmax 5 Curves Include Next gt Biacore T100 84 5 ES Kinetics Affinity Select Data Create Curves Curve Multiple Ligand N Sample 83 Temperature 37 C iv 10 Fc 2 1 15 11 Fc 2 1 15 7 Fc 3 1 15 8 Fc 3 1 15 9 Fc 3 1 15 10 Fc 3 1 15 11 Fc 3 1 15 Blank Subtracted Sensorgrams F Zoom lock Kinetics gt Fit ES Kinetics Affinity Fit Kinetics Create Curve Multiple Ligand N Sample 83 Temperature 37 C Add Fit Current Fits Model 1 1 Binding v EER D eesciptior Parameters i Delete J Pu Quality Control Report Residuals Parameters ka 1 Ms kd 1 s KD M 1 994E 5 1 948E 4 9 768E 10 2 219E 2 500E 8 Rmax 1 Cycle 8 Fc 2 1 2 77 nM i 2 77UE 9 Rmax
209. FLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLL LLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLL Tes J Startup Buffer l Yes 3 Sample Betazmicro 32 11800 Sample puc ee Betaemicro 8 11800 Yes 8 Sample Betazmicro 1 16 11800 F Ctrl T All sensnrgrams BEES Curve Name Fc 2 lt Assay Step Purpose Overlay P Cycle Overlay RU Sensorgram Zoom Lock Biacore T100 9 297 9 3 2 Sensorgram window _ cok Tools gt Color By Sample RU Sensorgram Zoom Lock 10700 a2micro high2 a2micro high1 a2micro low2 a2micro low1 9700 ZO Tools gt Report Point gt Id and Marker
210. SE Standard error Parameters SE SE 10 RI RI RI O Constan 1 ka Ka Rex
211. Te Open New Method Lookin 9 Biacore Methods Name Type Modified Ed Affinity in solution Method Builder 3 28 2008 Calibration Free Concentration Method Builder 3 28 2008 5 GST Kinetics Method Builder 3 28 2008 5 Inject and recover Method Builder 3 28 2008 5 Kinetics heterogeneous analyte Method Builder 3 28 2008 5 L1 liposome capture Method Builder 3 28 2008 D LMWw kinetics Method Builder 3 28 2008 5 LM screen Method Builder 3 28 2008 E NT kinetics Method Builder 3 28 2008 I Single cycle kinetics Method Builder 3 28 2008 Calibration Free Concentration Open Method Builder Main Overview 6 J Ki hi General Settings Biacore T100 5 149 T Method Builder Main Overview 1 t start General Settings gt Data collection rate Detection Sample compartment temperature Hz Vary with analysis temperature Assay Steps RC Cycle Types Variable Settings Miscellaneous 5 uffer settings Concentration unit M gt Posin Nm TN A
212. regeneration check blr ilz File Edit View Commands Run Tools Help bx ZEE 3 Cyde 1 Curve Sensorgram Fc 3 eue s3l0 H Lock scale i 2 lcs M 5 10 15 Fc Time Window AbsResp SD LASD Slope HelHesp Baseline Id Keywords in cycle 1 Value Flow 30 Flow Path 3 4 Online COM1 Temperature 25 00 9C Running manual run Sample compartment temperature current 25 C set 25 C Run Lime 1 min 4 2 View Show Only Current Curve 1 View Show All Curves View Show Curves of Same Type Inject command Menu bar Commands Inject Biacore T100 4 ZZP AEEA ABRA FROR NES 49 Inject Vial well position R2B1 amp s Minimum required volume in wiakwell For this injection B8 pl Vial well po
213. v Control Sample v Sample EUL Cycles ou Aa Id OK i Custom Report Points Custom Report Paints 1d Position Assay step purpose stabilityz n seconds after stap of Calibration injection Sample 1 Sample injection Control Sample Biacore T100 192 5 5 61 Toolbar P a Lau Bar Chart Bar Chart 345789101123 41 5I BL TI CGCP1220 32 42 Ee EAn 9E P3 CGORLOE 44 2034 4E TPALOTLOSCS 15 25 5 45 6 EGGS EBCB 16 25 24A p EG 6069 co e e e Relative Response RU binding x e HA e Cycle Humber Report Point Warfarine Warfarine Fc 4 3com ee Eee Warfarine Dm condition level condition level 2 condition baseline Warfarine zm conditian level 3 warfarine solvent 1 Warfarine 2e solvent 2 ier Warfarine EU SolventCorrectio Curve
214. 1 Cycle 9 Fc 2 1 0 33 nM 3 300E 10 8 330E 9 Rmax_1 Cycle 11 Fc 2 1 0 925 nM i 9 250E 10 Rmax_2 Cycle 7 Fc 3 1 25 nM 2 500E 8 a 2 Cycle 8 Fc 3 1 2 77 nM 2 770E 9 gt HT GSRI XnnGTulS Rmax Fit global Biacore T100 5 85 5 16 Y k X k Biacore T100 Kinetics Summary Tools Kinetics Summary Biacore T100 Kinetics Summary Thumbnails S Biacore T100 Kinetics Summary KineticsAffinity multiple Rmax bme File view Help ee Table Thumbnails On Off Rate Map Ligand Sample 83 Ligand Sample 83 200 0 200 400 600 800 1000 1200 1400 200 0 200 400 600 800 1000 1200 1400 Fit 1 1 Binding Fit 1 1 Binding ka 1 Ms 2101E 5 ka 1 Ms 1 742E 5 kd 17s 1 582E 4 kd 17s 1 809E 4 200 0 200 400 600 800 1000 1200 1400 200 0 200 400 600 800 1000 12
215. 10 C C i 148 Warfarine Sample Farine Sample i C e e e e C e e Startu p C e C C C C Co 148 buffer Startup 7 3 3 3 3 3 C C i1 G3 148 buffer Startup buffer Regeneratian ASL LALL IOL pe 5 DMSO Wash i C C C e C C C3 4151 50 DMSO Wash 4 Full Solvent correction Solvent correction buffer 3 e e e e e e C e Full i Solvent correction2 Solvent correction buffer 2 e e e e eo e Full i Solvent correction3 Solvent correction buffer A z e C C e C C e C3 Full Solvent correctiond Solvent correction buffer A 1 Full i Solvent corrections Solvent correction buffer e e e e e e d e i Full Solvent correction amp Solvent correction buffer v gt Bak Neo Cos U 5 72 Menu Export Positions
216. 11500 Online COM1 Temperature 25 00 9C Sensor chip CMS cn kan cn cn a cn c o co oon Sample compartment temperature current 25 C set 25 C Running standby remaining time 4 0 days l Biqcore T100 Evaluation Software Biacore T100 108 5 5 21 Run gt Stop Run Biacore T100 AN This will stop Ehe run stop Run Run Stopped Finishing curent cycle please wait Abort cycle hn Ctrl H Break _ Ctrl Breakl Biacore T100 Evaluation Software Biacore T100 5 109 5 2 2
217. 265 COMAC TUNC out eode 18 29 49 58 59 98 99 125 126 155 154 177 197 198 216 239 240 259 Control Sample gebipitioPiscaenadenenadein a a E A E A a A ER E UN reden 128 218 CONTO GM DIE NR mm 128 218 Gee GDN A E OEN 305 EAEE E E E L E E E E L E E E E E E 45 CUT OO 78 80 120 122 Custom Pet esan E AE P 35 CUSTOM REDOC FOM ranan a i i 141 191 12 NRE E E E a EEE A E E E 61 129 179 218 256 257 CUVEE TUDESaidesdatde adde vacimtoneind raciones 97 152 238 250 D Data 8o eile Rao E 95 149 236 251 DeboSsION SO UN EE EE EE SS 259 Deposition solutio volue icut easutes desideres tefte beside a ead eiie 259 Boo P 277 278 286 DesorD arid SANtZ Ei 278 286 BEE m e A E E EEE EEEE E T 58 95 125 149 175 196 215 236 251 259 PiS ocn inin r aA 5998 99 153 154 1715 197 216 259 DMSO U i 3 26 46 211 212 213 229 233 251 DOCKED iaaa NOTOTO OTONO OONO NOONA 6 276 289 E ED CONTENTER E TT 25 356 38 40 Erect RC 11 19 30 38 49 64 106 132 162 181 201 221 244 271 Elect ROCK TV 11 30 38 64 106 132 162 181 201 221 244 271 ED UU 281 287 End MANU TOM NN Ni 22 51 EARRING i 2225 EEEE E eT 125 175 260 Evaa torn Vande e 261 262 ESCUTE CU A 140 ENC UC 139 mee 227 EXDOLPOUIVOS Ee IL
218. 6 Predip Mix with Both 2 1 1 2 4 3 3 4 Detection Multi Fraction 20 20 80 Stabilization period after mix Mix Extra wash after injection with Stabilization period Capture
219. 7 4 266 9 Contact time 5 s Flowrate 30 p min Stabili S 2676 5 267 9 7 269 2 9 268 2 267 2 267 3 12 268 6 13 267 9 269 9 267 9 268 7 271 2 269 2 271 6 268 4 267 9 270 5 Relative response stability T e e e 15 Cycle number 20 Biacore T100 5 53 5 6 Kinetics Affinity 54 Concentration Analysis 123 Binding Analysis 174 Thermodynamics 193 Kinetics Affinity 211
220. Assay Step Purpose verlay Cycle Overlay ensorgram j All sensorgrams RU Sensorgram B9 Plot 3600 Baseline Sample Binding level Binding stability Binding to reference Report Point T able m Report Point Table C Zoom Lock Conditioning Sample Startup 300 Time 5 6 Keyword table Tools Keyword Table Keyword Table Cycle Assay step purpose Sample Conc nM Mw Da z o v Reset All Filters Af Conditioning Wu uM MIR 2 Startup buffer 3 Startup buffer 4 Startup buffer 5 Sample antigen 8 Sample antigen1 4 7 Sample antigen g 8 Sample f antigen 8 Sample antigen 10 Sample antigen 11 i Sample antigen 1 2 Sample antiqen1 1 E Sample antigen2 14 Sample antigen2 f M 1 5 Sample antigen2 1 amp Sample antigen2 1 7 Sample antigen2 18 Sample antigen2 1 g Sample antigen2 20 Sample antigen3 21 E Sample antigen3 22 Sample antigen3 23 Sample antigen3 24 Sample antigen3 25 Sample antigen3 26 Sample antigen3 27 Sample antigen3 28 Sample antigen3 Concentration Unit Biacore T100
221. Explorer Thermodynamics 2 Eg Thermodynamics antigen Biacore T100 5 211 5 7 BVJ _DMSO nM uM Ks 1 10 10 5 PBS HEPES HEPES DMS0O
222. Heterogeneous analyte Calibration free conc General Report Points Commands Report Points T E T E baseline 10 Before Start of Sample 1 5 Yes binding 5 Before End of 5Sample 1 5 Mn E w stability 10 After End of Sample 1 5 Mo E cn baseline 10 Before Start of Carry over control 1 5 Yes m ra binding 5 Before End af Carryv oaver control 1 5 Mo E co_stability 10 After End of Carry over control 1 5 Ma Name Sec Start of End of Inject Before After Start of End of Inject Biacore T100 Start of End of Inject Window Baseline 6 263 Iniect
223. Lmesnd ccc iritisl binding Sample MW 156000 i Sampe 1 Sample E553 1 ms Tema 25 Temp rature 25 00 9c Sample compartment temperature currenk 25 C aet 25 C l Biacore T100 Evaluation Software Biacore T100 164 5 5 44 Run Stop Run Hiarnre T100 AN This will stop the run stopRun Run Stopped Finishing curent cycle please wait Abort cycle hn Ctri Break _ Ctrl Breakl _ Biacore T100 Evaluation Software Biacore T100 5 165 5 4 2 Evaluation
224. M uu Eu 95 149 256 247 250 B social gc e 51 scie mete M 21 67 110 135 166 184 204 225 232 263 294 SOS RARIOR REIECTA NE NETTES 68 81 88 Bidcore Maintenance MT H 275 277 278 285 285 286 Binding level I 67 110 135 166 184 185 204 225 294 PIRAN TOTT T E OP cM eM MM ULL E 67 110 135 166 184 190 204 225 232 294 Bilent AIO EB RNN 000000000000000000000 VNVONVANNNNNAAUO000000000000000020008000MUMMD0U OU 72 115 BlanKIMMODIIZOUON i i i i i 35 BOUN m 40 EVIE EE LRE cuoc vM EIE III ELIMINA T TT 95 149 236 252 SAEI S EARS To NEN NU TEES 45 C calibrato CUNE cm 127 COND O T ELSE T27 COPPE NN 58 125 175 196 215 259 260 CONV OVET aa i 58 196 215 CONV 9 Ni i i i i i 232 261 CCN E 143 75 76 91 118 Fete iq igojio n TETTE 53 60 95 124 127 136 138 141 149 199 217 236 252 262 Concentrauonp ATDlVSlS onset tla i a fi Ni 53 124 136 138 141 Concent atona i 95 149 236 252 Biacore T100 Concentrations per CVele sostenible dd hated dti iet dd ene hti etd n UD D dh D Oh D Bd heb Bde 98 Kee Te TATUR 1o 2 75 58 125 175 197 215 240 254 Sea emersuu u cm T 254
225. Method Te Open New Method Look in E Methods nd Templates Method Builder 4 2 2008 Method Builder 3 13 2007 Methods and Templates 2 Browse Open Biacore T100 6 249 T Method Builder Main 2 Assay steps General settings Concentration unit nM General Settings Startup Data collection rate 10Hz E Startup LMW kinetics 3 times as entered E les Eier Detection Dual Assay Steps ri Sample Cycle Types Sample LMW kinetics 1 time as entered Settings for assay step Startup Temperature 25 C Variable Settings Solvent correction Buffer t Solvent correction Solvent correction 1 time as entered Before after every Control sample t Control sample LMW kinetics 1 time as entered Before after every Settings for cycle type LMW kinetics Sample 1 varies by cycle 60s 600s Carry over control 1 i Report points Expand ll Collapse All
226. P3 m a 11 Control 118 Control 118 Control 118 Control 118 Control 118 Control 118 Control 118 Control 118 1 118 10 118 11 118 12 118 13 118 14 118 15 118 16 118 17 118 18 118 19 118 2 118 20 118 21 118 22 118 23 118 24 118 25 118 26 118 27 118 28 118 29 5 179 0 7 LN ges ii Control sample Control sample Control sample Control sample Control sample Control sample Control sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample v ul Biacore T100 180 5 Menu Export Positions
227. PA Kinetics Linked Reactions Kinetics Mass Transfer E Assay Kinetics Affinity PA Binding Analysis Concentration Analysis Thermodynamics Look in fa Methods nd Templates Name 3070814 3070913 3080314 ue Lowe Surface Preparation Immobilization New Methods and Templates 2 Open Browse Open Te Immobilization Immobilization Setup Chip type CM5 Flow cell 1 Immobilize flow cell 1 Flow cell 2 nE Flow cell 3 Immobilize flow cell 2 Immobilize flow cell 3 Z Immobilie flow cell 4 Biacore T100 le 3 35 Chip type CM5 Flow cell 4 Irme la lem zc Method 3 Aim for immobilized level Ligand Protrein amp 20ug ml pH5 Dilute liga
228. Pooling Yes OK Automatic Positioning Biacore T100 5 201 Eject Rack Rack tray port OK Eject Rack Tray Rack Positions Next gt 9 Te Thermodynamics Prepare Run Protocol Tahoma 1n Prepare Run Protocol Make sure the correct sensor chip is docked Make sure all samples amp reagents are loaded in the rack and microplate according ta the Rack Positions setup vials should be sealed with rubber caps and microplate with adhesive foil Flace the bufferis on the left hand tray and insert the correct tubing s see below Mote Standby after run will use buffer A Make sure there is fresh water in the water bottle on the right hand tray If necessary empty the waste battle before start of the run Estimated run time 12h 13min excluding conditional statements temperature changes and standby Flow Estimated buffer consumptian 3 J
229. Q Single hade Batch mode Evaluation settings Evaluation purpose Kinetics Samples Curve type ReferenceSubtracted v Include Curve Ligand Sample v Fc 2 1 antibody 10ug ml pH5 v Fc 2 1 antibody 10ug ml pH5 v Fc 2 1 antibody 10ug ml pH5 antigeni antigen2 antigen3 Check All Uncheck Al Samples Evaluation purpose Model Kinetics Affinity Calculating Results Samples Fitting sample 1 of 3 antibody 1Hunrml pH5 antibody 1Bug ml pH5 antibody 10ug ml nH5 Tem s antigeni antigenz antigen3 Iteration 7 Chr 0 307 RUF Mas relative change 4 507 RIit 2 0 8337 Accept Curent Abort Current Abort Remaining Biacore T100 82 5 Biacore T100 Evaluation Software Kinetics antigen antibody blr tew Evaluation Tools Window Help Plot ai Bar Chart S m lel Sensorgram AJ Kinetics Affinity v Concentration Analysis v Thermodynamics a senic o cdd antigen1 TER AEE BAA AEEY x Remove v Edit Curve Fc 2 1 Ligand antibody 10ug ml pH5 E Curve Fc 2 1 Ligand antibody 10ug ml pH5 Curve Fc 2 1 Ligand antibody 10ug ml pH5 F Sensorgram IR Al sensorgrams Fits 1 1
230. Sample i 148 Analyte A Sample e e Q Q Q Q C 148 Analyte A Sample o Q Q O Q 148 Analyte A Sample Y Q Q Q O C O 148 Analyte A Sample i 148 Analyte A Sample 148 Analyte A Sample 148 Analyte A Sample 148 Analyte A Sample eMe Back Next gt Menu 7j 5 Automatic Positioning amp 3385 Te Automatic Positioning Change the order in which the samples are positioned by ordering the regions The first region in the list is positioned first Region Color Orientation Anchor Rack Vial Si i First Sort By Control sample E Cyan Column Bottom left Sample Small i Ascending Sample E CarkElue Column Bottom left Sample Small i Ascending Startup I crimson Column Bottom left Sample Small Ascending Wash Yelaw Column Bottom left Reagent Large Content Ascending Solvent correction buffer amp Il Blue Column Bottom left Reagent Small J Content Ascending gt ol Auto Pooling_ Yes OK Automatic Positioning
231. Transfer Ready Sensorgram ize 52 Wan Sut Event log 85 7 Wash Ready 953 Transfer Start 52 pl R2B2 R2B3 d 40000 1389 0 Transfer Ready 1380 Mix Start 111 pl R2B3 WI n OW 38000 251 3 Mix Ready 285 8 Inject Start R2B3 705 8 Inject Ready 70 yl 36000 719 9 wash Start 750 4 wash Ready 750 4 Wash R2B4 oen 8053 Wash Ready 840 5 Inject Start R2B5 32000 1260 5 Inject Ready 70 pl 0 200 400 600 800 1000 1200 1400 12735 Wash Start Time 1304 3 Wash Ready 2 E 13382 Inject Start R2B4 Re po rt po nt Fc Time Window AbsResp SD LASD Slope RelResp Baseline Id Keywo 1753 2 Inject Ready 70 yl 4 2D 5 359025 007 007 0 02 00 Yes Baselne ERE 17723 Wash Start 5 350287 010 004 0 05 1261 No EDC NHS Contaci 18023 Wash Ready 4 0 5 374125 088 003 047 15099 No Ligand FlowRaj 18180 Temperature 2500 C ta b e 4 1813 0 5 37455 0 40 0 04 021 15133 No Immobilized ij Ligand eni SU pH Method Am ne n Procedure Time amp ndFlow Keyword table Online COMI Temperature 25 00 9C Running method Status bar Sample comfffirtment temperat NT 40 C set 40 C Run time 6 h 55 min Estimated run time 15 h 7 min Online COMI Temperature 25 00 c Running method Sample compartment temperature current 40 C set 40 C Run time amp h 55 min Estimated run time 15 h 7 min Menu bar Biacore T100
232. Ut gi Immobilization Assay Development Regeneration Scouting Buffer Scouting Surface Perfarmance Control Experiments Kinetics Linked Reactions Kinetics Mass Transfer A Sa Kinetics Affinity Binding amp nalysis Concentration Analysis Thermodynamics Surface Preparation Immobilization pH Scouting New Methods and Templates 2 Open Browse Open Te Immobilization pH Scouting Setup Detection Flow path Buffers Buffer Name 10 mM Acetate 10 mM Acekate 1 mM Acetate 10 mM Acetate Flow path Next Biacore T100 3 29 Te Immobilization pH Scouting Injection Parameters Ligand Sol
233. every 1 Calibration points Concentration 6 2 Jg Next gt Biacore T100 128 5 Te Concentration Analysis Control Samples Control sample definitian Run control samples Repeat control zample s every sample cycles Control samples Expected conc Control Sample id Control Sample definition Run control Samples Repeat control Sample s every 20 Control Samples Control Sample id Expected conc Next gt T Concentration Analysis Samples Sample table Sample id Dilution factor 501 2436 01 50
234. jj Approximately 200 ml z IURBVGIIE mIR WERS DEERLDLTIL Z Z amp DARSe7 mof Start Save as Methods and Templates 7 Bia Users Don t Save Biacore T100 182 5 3 regeneration check blr regeneration check wizard blr surface parformance wizard blr Thermodynamics antibody vs antigen Save in File name Sqve l REI Eld Standby flow l Biacore T100 Evaluation Software DB HE 5 55 P PIN 3 din Run gt Stop Run Hiacnre T100 AN This will stop the run stop Run Run Stopped Finishing curent cycle please wait Abort cycle hn Ctri Break
235. standard value 1 007 to water at 20 C Enter walue I I I l I l caLcurnaTE Diffusion coefficient at 20 C AILO REA mis i k ALL Convert diffusion coefficient from temperature T to 20 C Temperature T 25 iie D at temperature T 4 00e 11 imas CONVERT Diffusion coefficient at 20 C 3 49 11 im s 20C D Molecular weight 7358 Da Friction ratio FRK Choose molecular shape F I Y 2 AT Globular 1 2 DYVE amp JYYVYVILTZZYDBA 3 e Globular 1 2 BI Moderately elongated 1 7 e Elongated 25 I8 E amp UV97 V28 O Enter value
236. time 4h 238 min escluding conditional statements temperature changes and standby Fow Estimated buffer consumption f Running buffer l Approximately 200 ml Cx ss Start Save as Methods and Templates 7 Bia Users Don t Save Biacore T100 5 65 Save Results From Run s Savein T100manual Immobilization of antibody blr Immobilization of Prteina blr manual blr 3 pHscouting blr 3 regeneration check blr regeneration check wizard blr surface parformance wizard blr Thermodynamics antibody vs antigen blr File name Kinetics antigen antibody v Save as type Result file blr v Save in File name Sqve Standby flow l
237. window Export Curves txt Excel Biacore T100 306 9 text Excel File Export Result To Excel xls Evaluation Explorer Excel xls Beta2micro hi Beta2micro high2 X Beta2micro high2 Y oi RPoint Y 19 0 55859 3 0 071289 19 0 833984 3 0 0576172 18 0 533203 0 0 18 0 761719 0 0 18 0 74F 17 0 5625 2 0 0820313 17 0 779297 2 0 102539 17 16 0 457031 182 332 89 16 0 740234 182 332 645 16 0 60 15 0 417969 185 333 021 15 0 712891 18
238. 0 pA X E 14 S AULUS L 249 b pv GC MR A 274 275 276 286 289 b qu ios o ETERNI 276 Biacore T100 ii 00 T M I 26 46 211 BRE S LM LM MM E MM 45 73 75 91 116 117 124 2I RM 197 212 213 226 228 229 257 261 264 2 213 226 227 228 229 255 257 264 A E A 229 as arb IE EB OBRA cu b d RR OR EO baeo Fano nei aes 228 MMC HEP HE De DI I 185 187 189 230 PIT E T OE e 9 BUSH TR as ei uem e sede c e dc std D 3 T A 14 23 24 25 26 27 UITAE IV RE TT 25 28 J HY KAIRO DEOR US c oet ona data data ate dat ucl aor e die 27 Pur dbi wustwP 51 35 45 47 67 73 110 116 124 125 135 166 184 190 204 212 213 225 229 232 294 ys iyd s pium a RR EE AA E T ONAA TA 20 21 50 51 Lob a a Due ARAM E UCM AL LR LM S 20 21 22 50 67297 Ioco AS EDO Tecta A AA oe todos ot 20 141 191 262 ETT OST m 54 176 Biacore T100
239. 00 1400 1 1 Binding Fit 1 1 Binding Table Include Table Thumbnail On Off Rate Map emus smeelewvelrmeco f T a OE wm v KineticsAFFinity multiple Rmax Fr 3 1 1 1 Binding z 101E c5 1 692E 4 8 0SSE 10 w KineticsAFFiniby multiple Emax 89 2 59 Fr 4 1 37 1 1 Binding 1 742E 5 1 809E 4 1 038E 9 wv KineticsAFFiniby multiple Emax at Fc 3 1 37 1 1 Binding 1 996E c5 1 779E 4 8 909E 10 v KineticsAFFinibv multiple Emax 90 2 at Fr 4 1 37 1 l Binding 1 65675E 45 1 867E 4 1 115E 9 v KinekiceAFFiniby multiple Emax 91 g1 Fr 3 1 37 1 1 Binding 5 721E 4 4 8 236E 4 1 440E 8 v KineticsAFFiniby multiple Rmax 912 a Fc 4 1 37 1 1 Binding 1975 3 7489E 5 1 898E 8 Biacore T100 86 5 On Off Rate Map S Biacnre T100 Kinetics Summary KineticsAffinity multiple Rmax bme Fie view Help AEA m 37 Table Thumbnails On Off Rate Map Selected Evaluation Ligand Sample 30 30 100000 8 Fi E 8 200 0 200 400 600 800 1000 1200 1400 Fit 1 1 Binding ka 1 Ms 1 996E 5 kd 17s 1 779E 4 1e 4 Off rate kd log tem Ligand Sample Curve Temp C FE ka kd KD 89 89 Ki
240. 00 ul 100 96 DMSO 400 ul 10000 ul 6 DMSO A F v 27858 1x 27 V7 TREA 9400 ul 100 96 DMSO 600 pl 10000 ul 4 6 DMSO 8 DMSO 496 DMSO 100 200 500 400 500 600 700 6 DMSO 700 600 500 400 300 200 100 700 700 700 700 700 700 700 700 ul Biacore T100 214 5 5 7 1 Toolbar Run Wizard Menu bar Run gt Wizard 2s Te Run Wizard Template Lookin Methods nd Templates Name 070814 3070813 3080314 30805 orz zzo pun i t EMO 5 5 SS LEET IPE g mo c 0280 ozo Bgoso9 5s29Gm EZ o o 0830 oO Dm zz 9 79859325 EE T 222 ZSD Q oz o 0 ERES 2 922320525523 55z ER 2 2 S S5 D 2 ez 3 E 5 o O D 2 2 A 292 z e ww g 1 A J Con A v Ass A A A PA Assay Kinetics Affinity New Methods and Templates 2
241. 00to 1400 RU 5010 50 RU B Refractometer Biatest solution Baseline level 22715 21400to 23600 RU 35922 22691 21400 to 23600 RU 35427 22514 21400to 23600 RU 35574 22550 21400to 23600 RU 36032 Average 22618 35739 Variation 201 2600 RU OK 605 23000 RU OK C Injections Rise Fall Fc1 1090 30 OK 0 25 96 OK Fc2 100 90 OK 0 95 96 OK Fc3 100 7250 95 OK 0 5 96 OK Fc4 100 290 9 OK 0 lt 5 OK Leaks Fct E 2100 RU OK Fc2 16 2100 RU OK Fc3 10 2100 RU OK Fog 2 2100 RU OK OK BAD BAD Biacore T100 8 289 8 4 4 8 1 Standby flow A
242. 1 2436 01 501 2436 01 501 2436 01 501 2436 02 501 2436 02 501 2437 01 501 2437 01 501 2438 01 501 2438 01 501 2439 01 501 2439 01 501 2440 01 501 2440 01 505 0001 01 505 0001 01 TT g Biacore T100 5 129 Sample id 977 oO Dilution factor Next gt i Excel Excel txt Te Concentration Analysis System Preparations Frime before run Normalize detector Temperature settings Analysis temperature C Sample compartment temperature PC ie Cenn Prime Normalize Analysis temperature 25 C Sample compartment temperature 25 C Cycle Run List Te Concentration Analysis Cycle run list Assay stepname Sample 1 Solution Sample 1 Conc ng ml
243. 1 Binding Fits 1 1 1 Binding Fits 1 1 1 Binding v g Plot Baseline Sample Binding level Binding stability Binding to reference Report Point T able rj Report Point T able Kinetics Affinity J antigenl l A antigen2 AJ antigen3 f 10 5 100 50 0 50 100 150 200 250 300 350 100 50 0 50 100 150 200 250 300 350 100 50 0 50 100 150 200 250 300 350 Time_ s Time s Time s PEA Quality Control Report Residuals Parameters Quality Control Report Residuals Parameters Quality Control Report Residuals Parameters Kinetic constants are within instrument Kinetic constants are within instrument Kinetic constants are within instrument specifications specifications specifications Kinetic constants appear to be uniquely Kinetic constants appear to be uniquely Kinetic constants appear to be uniquely determined determined determined o No significant bulk contributions RI found o No significant bulk contributions RI found o No significant bulk contributions RI found Check that sensorgrams have sufficient Check that sensorgrams have sufficient Check that sensorgrams have sufficient curvature curvature curvature Examine the residual plot Pay attention to Examine the residual plot Pay attention to Examine the residual plot Pay attention to systematic and non random deviations systematic and non random deviations systematic and non random deviations
244. 300 ml EUCKRBVGOERESIR AERA DERIVI YIREREN RRENA Start Save as Methods and Templates 7 Bia Users Don t Save Save Results From Run hs Save in 3 T100manual Immobilization of antibody bir Immobilization of Prteina blr My Recent manual blr Documents pHscouting blr regeneration check blr regeneration check wizard blr surface parFormance_wizard hlr My Documents da My Computer e File name Thermodynamics antibody vs antigen My Network Saveastype Result file bl vi Biacore T100 222 5 Save in File nime Sqve l REI Eld Standby flow l Biacore T100 Evaluation Software 5 74 Run gt Stop Run
245. 33 238 239 240 246 261 m T MEE RN OTOOTO 246 Te p CT 233 239 Biacore T100 PR 80 122 HE NEUE Ea eto 24 45 53 54 55 56 75 91 93 117 193 194 205 209 211 224 230 259 DL RU ass TS OA D LL MM M INL M EE M ME 55 66 78 109 120 jdn DLE uu D E E A A 54 55 56 93 peo 75 91 118 KE 143 145 IR SUL Es a r md PERENNE OE E E E 145 Li 305 A 223 232 260 a I RA AA TE E A ET 39 65 108 133 164 182 202 222 245 273 lt 192 RIPE RR 45 277 zk 53 174 230 231 cue EE ao RE RR 187 kc 24 123 127 136 137 139 140 254 imis 320 RIZ BE eccaucustnau onu op enano nat nano eta ote au e ud 143 Tant 7 8 14 23 24 25 26 27 28 29 32 33 34 35 37 38 39 40 41 42 43 44 DPI 44 EE EA E 24 33 35 39 40 41 42 43 44 83 84 232 DEREDE a i cn 40 EE 4 3 9 128 141 177 178 185 186 187 190 217 218 232 254 255 257 290 L CC 77 78 79 119 120 121 E e cue eM eM ML MM M MM Cu MEL 253 254 258 264 T c 45 49 50 51 52 54 Ec oguueccuqnc ca Ms LL IM EU E E CU 45 46 50 52 53 233 FEAESEPECDRS oed Epod be let abu mud Leti p
246. 37 M NaCl 596 DMSO pH7 4 HBS EP 200 ml BR 1001 88 0 01M HEPES 0 15 M NaCl 3 mM EDTA 0 005 96 v v Surfactant P 20 pH7 4 HBS P 200 ml BR 1003 68 0 01 M HEPES 0 15 M NaCl 0 005 96 Surfactant P 20 pH7 4 HBS N 200 ml BR 1003 69 0 01 M HEPES 0 15 M NaCI pH7 4 RREZE fESIARODZLSBIdUIBEC dDOD E Cad DEIA 0 22um JUL 9 C 538 d5 73 2 Biacore T100 4 1 1 1 3 Start 72 5 All programs Biacore Biacore T100 Control Software Te Biacore T100 Control Software Immobilization of Prtein blr ill File Edit view Run Tools Help S n m m y 839 Cycle 1 Curve Sensorgram Fc 4 T Biacore T100 Control S04 e Immobilization of Prtein blr DAR Menu ba r m Edit View R Tools Help i E E lian EN z 5 79 i Cyce 1 v Curve Sensorgram Fc 4 ProtreinA 20ugi ml pH5 Event Log Time Information 2000 0 0 Timestamp 5 8 2008 1 38 13 PM Tool ba 50000 0 0 Temperature 25 00 C 7h 4 I 0 0 Data collection 1 Hz Event log 71N 0 0 SetSample Temp 25 C 48000 0 0 Use Buffer A 0 0 Actual Sample Temp 25 C 0 0 Flow 10 l min 46000 0 0 SetTemperature 25 C 0 0 Flow cell 4 44000 0 0 Degasser On l 11 2 Tia pe 62 yl R2B1 R2B3 552
247. 5 332 791 15 0 53 14 0 411133 187 332 999 14 0 701172 187 332 898 14 0 501 13 0 400391 197 327 708 13 0 618164 197 327 761 13 0 421 12 0 310547 200 325 22 12 0 567383 200 325 211 12 0 35 11 0 216797 202 323 455 11 0 496094 202 323 496 11 0 45 10 0 198242 10 0 349609 10 0 431 9 0 182617 9 0 414063 9 0 33 8 0 25293 8 0 391602 8 0 436523 7 0 181641 7 0 267578 7 0 291 6 0 143555 6 0 24707 6 0 204102 5 0 0888672 5 0 15332 5 0 126953 4 0 178711 4 0 170898 4 0 122 3 0 0712891 3 0 0576172 3 0 09F 2 0 0488281 2 0 0605469 2 0 0 0 0195313 1 0 i 0 0488281 0 0 0 0 0 0 0283203 0 0517578 0 11 2 0 0820313 2 0 102539 2 ES 3 0 189922 3 0 194336 3 0 1 4 0 130859 4 0 303711 4 p TE ap B 0 Eyaluation WKinetics Affinity Name Beta2micro Sample Beta2micro Temperatu 25 Curve Fc 2 1 Model 1 1 Binding Description Curve Cycle 5 2 nM Cycle 6 4 nM Cycle 8 nM Cycle 8 16 nM Cycle 9 32 nM Cycle 10 8 nM ka 9 Ms kd s KD M 1147601 0 002635 2 3E 09 24 50454 Rmax RU Conc M tc 1 17E 08 2E 09 AE 09 8E 098 1 6E 08 3 2E 08 8E 09 Flow amp ul m kt RU Ms RI RUD 30 3 63E 09 30 3 63E 09 30 3 63E 09 30 3 63E 09 30 3 63E 09 30 3 63E 09 Biacore T100 0 178485 0 31193 0 113137 0 242235 0 992655 0 212286 36 M 4 MINFile Properties Baseline Sample Binding level B
248. 6 5 18 42335 5 19 42335 1 20 42334 7 21 42334 3 22 42333 8 23 42333 4 24i 4 E mi Assay Binding to referen ce 15 20 25 Cycle number 5 233 5 8 1 2 ul 1 1
249. 600 Sample 150000 5 09E 11 i 118 mouse IgG x1 1600 Sample 150000 5 09E 11 61 mouse IgG x1 400 ample 150000 5 09E 11 ow O Ow C i 118 mouse IgG x1 400 ample 150000 5 09E 11 61 mouse IgG x1 100 ample 150000 5 09E 11 O O X C 118 mouselgG x1 100 Sample 150000 5 09E 11 O CO O xs ard pH1 5 xum oo O GOIA SOI 2990 EEL OQ 00 BOON o9 oeoe C 2 2 2 2z 2z 2z 2 Z lt i oo OO mm NN D E 5 42 Menu Export Positions DEFALAS Menu Simple Position Import
250. 6E 6 5 790E 12 7 145E4 7 30 00 2 2ZUE 8 30 00 2 2ZUE 8 30 00 30 00 30 00 BATTU ISd Ncof sIid REELT Current Fits Current Fits Finish Biacore T100 5 121 F Kinetics Affinity Fit Kinetics Create Curve Fc 2 1 Ligand antibody 1 ug ml pH5 Sample antigeni Temperature 25 C Add Fit Current Fits Model 1 1 Binding 1 1 1 Binding Description Delete c 1 531E 6 0 002610 1 705E 9 105 3 7 178E 7 1 100E 9 2 231E 8 0 05105 2 200E 9 2 231bE 8 0 2011 4 300E 9 2 231E 8 0 1965 8 500E 9 2 231E 8 0 1167 1 700E 8 2 231E 8 0 1062 f V2 24 9 Rmax R Add Fit Parameters Parameter Settings Two State Reaction Fit Initial value Fit global 1e5 Default Fit global 1e 2 Default Fit global le Default Fit global le 3 Default Fit global YMax Default Fit global 1e8 Default Fit local Maxj5 Default Rir
251. 7 Bia Users Don t Save Save Results From Run As Saye in T10 0manual Immobilization of PrteinA blr manual blr pHscouting blr regeneration check blr My Recent Documents 3 Desktop My Documents 9 My Computer z My Network Save _in File name Sqave Biacore T100 regeneration check wizard blr surface parFormance wizard blr File name CFCA i Cancel Save as type Result file blr b 5 163 Te Biacore T100 Contral Software CFCA Example 1 Bil IEG ne Edt mem Rm Touk Hep 8 X d Eki fer cm d zy n E Cycle re Sensargram Fc L O Lock scale f Sart A101 Dijzcciaon Ready dabiiy t Sion Imet Sat Foz Resty 50 ul Wist Fired a Tepesture 25 0 C 250 Base dd Keweoudsiicecke 3 Fel Value Yes basare AstapSlep Ab anpe Me initial bind AssaySlepPurpose Ab Sampi binding CycleTupe Ab sampe EE Sample 1 Blank n baee Ssmpie 1 D l 4E iritisl binding Sample 1 lieu rae ln binding Sample 1 Ligard i Sample 1 Luogend cox Earalrg Sampe
252. 7 Toolbar Start Manual run Ep Menu bar Run Manual run 7 Te Manual Run Flow Flow rate 30 ul min Flow path Reference Detection in How cell s 3 4 subtraction D E Flow path 1 O e Flow path 1 2 O EI Flow path 2 iO EJ Flow path 3 4 Q a Flow path 3 Q Flow path 1 2 3 4 Q EI Flaw path 4 Flow rate 30 ul min Flow path Reference subtraction 2 1 4 3 2 1 3 1 4 1 Rack Start Save Results From Hun As Savein 3 T100manual O BEI 7 Immobilization of Prteina blr amp manual blr My Recent pHscauting blr Documents Desktop P Mu Documents da hy Computer File name regeneration check v My Network Saveastype Result file bl v Filename Save Biacore T100 48 4 T Biacore T100 Control Software
253. 86 Reagentrack TV aaa aaa A 12 PAG rack I a E E E O OONO I O MA C 12 Recovey Roto T T 259 RO CT NE ME UE CC 254 255 257 265 Relerence llle ee de tdt ASA aE E Sr SNAS 20 Relerehee LINE aana OU G 20 51 RegernierabllOrn ucscaa cese eA 50 52 58 59 98 99 125 126 153 154 175 177 196 198 215 240 259 260 ReOeherdl or scOUNNG RT i i i 52 RENOVE SeleEU ORR 70 113 RDGOMOSSOV3S0GDWWIK i 255 Repeatcdibratioh eveV RN ER E E ER E 127 Repeat Control Sdmiple 09 EV Lus hosscutind isi teh ond adt RN 178 Biacore T100 REDOT bas 4 21 75 117 136 142 189 230 262 297 298 299 Repor COIN ananena Ri 4 21 136 298 299 Report Pont Td 189 Re OT Tt 55 90 usse SE E 74 76 117 118 xs Morro M ii i i 306 Rense Ch M 5 RC i 13 1516 7 79116 1175 118 10 121 212 RS NET TTE 20 550655 I HT bo oS 01 91 92 116 117 119 121 S Sampe ana re dge Nt EACK m T 12 Sample compartment temperature sss 29 37 61 95 103 129 149 178 197 218 236 241 251 ER 76 119 SASE AAS E AS AE NAO 274 275 276 Sensorgramo3AdIUSLTie ib uae tens EEAO Ni 298 300 301 POWINNI URUK 48 Show overrode DONK CSS Sirasa A A 68 88 111 SMO CUMS O ME TV Ch 48 Show Only COTENT C UV E sev onore on ipn va Ni Ni Ni hn 48 SIRO M b MEM bM d MM EM 68 88 205 Singiescucle SIR unninn CG ee 94 148 SOVE
254. ATION 1 1 Biacore T100 5 175 Detection Flow path 2 1 4 3 Chip Chip type Capture 2 4 Sample 5 40 Enhancement 2 Regeneration 1 2 Next gt Te Binding Analysis Setup Conditioning Bun conditioning cycle Contact time ES s Number of injections Startup Run startup cycles Solution Mumber of cudes 3
255. Analyte A Sample i 148 Analyte A Sample e e Q Q Q Q C 148 Analyte A Sample o Q Q O Q 148 Analyte A Sample Y Q Q amp Q O C O 148 Analyte A Sample i 148 Analyte A Sample 148 Analyte A Sample 148 Analyte A Sample 148 Analyte A Sample eMe Back Next gt Menu 7j 5 Automatic Positioning amp 338 5v Te Automatic Positioning Change the order in which the samples are positioned by ordering the regions The first region in the list is positioned first Region Color Orientation Anchor Rack Vial Si i First Sort By Control sample E Cyan Column Bottom left Sample Small i Ascending Sample E CarkElue Column Bottom left Sample Small i Ascending Startup I crimson Column Bottom left Sample Small Ascending Wash Yelaw Column Bottom left Reagent Large Content Ascending Solvent correction buffer amp Il Blue Column Bottom left Reagent Small J Content Ascending gt ol Auto Pooling_ Yes OK Automatic Positioning
256. Biacore T100 5 159 LS Method Builder Variahles Assay steps Blank Flow rate Ll min XR Sample Next gt Assay stepname Sample 1 Solution Sample 1 Flow rate pl min Sample 1 MW Da Sample 1 D 20 buffer buffer buffer Buffer Buffer mouse IgG x1 6400 150000 5 09E 11 mouse IgG x1 6400 150000 5 09E 11 mouse IgG x1 1600 150000 5 9E 11 mouse IgG x1 1600 150000 5 09E 11 Buffer Buffer mouse IgG x1 400 150000 5 09E 11 mouse IgG x1 400 150000 5 09E 11 mouse IgG x1 100 150000 5 09E 11 mouse IgG x1 100 150000 5 09E 11 Biacore T100 160 5 Next gt T Method Builder System Preparations Prime before run Normalize detector Prime Normalize Next gt Te Method Builder Rack Positions MW Da 20 C Fe b oc 61 Buffer Sample S i 118 Buffer ample 61 Buffer 118 Buffer 61 mouse IgG x1 6400 ample 150000 5 09E 11 118 mouse IgG x1 6400 Sample 150000 5 09E 11 61 mouseIgG x1 1
257. Bl desarb Solution 2 an the night hand tray and insert Ehe water inlet tube BlAdesorb Solution 2 25 ml 15 ml 2 A BC D BlAdesorb Solution 2 25m BIAdesorb Solution 2 15 ml Start 2 3 liEsnrh and Sanitize Step 3 Wipe the pump inlet tubes with a moist tissue Place 50 ml diluted Bl amp disinfectant Solution an the left hand tray and insert all four pump inlet tubes Place 30 ml diluted Bl amp disinfectant Solution an the right hand tray and insert the water inlet tube BlAdisinfectant Solution 50 ml 30 ml 2 A B C D BIAdisinfectqnt Solution 50 m BIAdisinfectant Solution 30 m Start o Biacore T100 280 7 3 4 Desorb and Sanitize fx Step 4 Wipe the pump inlet tubes with a moist tissue Place water on the left hand tray and i
258. Curve Fc 2 1 Ligand antibody 1 ug ml pH5 Sample antigeni Temperature 25 C Add Fit Current Fits Model 1 1 Binding v 1 1 1 Binding Description Delete c 1 531E 6 0 002610 1 705E 9 105 3 7 178E 7 1 100E 9 2 200E 9 4 300E 9 8 500E 9 1 700E 8 2 231E 8 0 05105 2 231bE 8 0 2011 2 231E 8 0 1965 2 231E 8 0 1167 2 231E 8 0 1062 Rmax R Add Fit Parameters Parameter Settings Two State Reaction Fit Initial value Fit global 1e5 Fit global e 2 Fit global 1e 3 Fit global le 3 Fit global i YMax Fit global 1e8 Fit local YMaxi5 Default Default Default Default Default Default Default 9 Cra Rmo Rr Fit Fit local 0 RI RI Fit
259. DIMJECT Specified in Immobilization Setup 4 INJECT Ethanolamine 420 10 OK Biacore T100 3 37 Tes Immobilization System Preparations Frime before run Normalize detector Temperature settings Analysis temperature C Sample compartment temperature C Prime Normalize Temperature settings e Analysis temperature 25 C Sample compartment temperature 25 C Next gt e immobilization Rack Positions uM 89 EDC Immob Fc 4 89 MHS Immob Fc 4 Empty EDCINHS min capacity 124l Immnh Fc 4 129 Ethanalamine Immoab Fc 4 a8 Pratrein 2 ug ml pH5 Immnh Fc 4 U Biacore T100 38 3 EDC 89 ul 7 mm NHS 89 pl 7 mm NHS EDC 7 mm Ethanolamine 129 ul 7 mm
260. Define some values in Method and others at run time 5 73 OO Bs REI E CANZJU o fiot amp ss IRE OGB BEC AZJ Verification Biacore T100 5 101 T Method Builder Main Verification results The method has been verified and can be used to set up a run General Settings Assay Steps Cycle Types Variable Settings Verication The Method has been verified and can be used to set up arun Setup Run Te Method Builder Detection Detection Elow path s Flow path Next gt Biacore T100 102 5 Startup Sample T Method Builder Variables Startup Sample Variable values for amp ssay Step Sample sample solution Conc 1 n Conc 2 nM Cone 9 nM Conc 4 9 Conc 5 W CDa E Sample1 Sample Solution Conc nM MW Da Conc 1 Conc 5
261. E Cyan E CarkElue Startup BI crimson Wash E vellow Solvent correction buffer amp Il Blue Control sample Sample Column E Column Column Column Column Bottom left Bottom left Bottom left Bottom left Bottom left Sample Small Sample Sample Reagent Reagent Small Small Large Small Y Y Y Pooling Auto Automatic Positioning Ascending Ascending Ascending Ascending Content Ascending gt 9 ess Pooling Ves OK Biacore T100 105 106 5 Eject Rack Rack tray port l OK Eject Rack Tray
262. GE Healthcare 9 Biacore Life Sciences Biacore T100 Ver 2 Instrument Handbook GE imagination at work MELOS TS Gc Ec 1 1 1 1 trL TEOLOE ERR RETE ERROR ERI EORR 1 1 1 2 DYIY TREA KOC Y IS kk 2 1 1 3 4 1 2 kk 5 1 2 1 zd i kk 5 122 0 OJAT Y ETRE SIEG Ime atem tSc oi Gc 9 joo E Ed nuu I EAMU EM IEEE UM EEUU EM EUM IUE EM EE 10 EE ODEA A LD Fa ri 11 JE 1 U 14 2 1 a A IP ODE a D a Dy KK 15 SIME s 1 7 18 2 12 199 ve T aA ADDEN I 20 2 1 3 VODI e E 22 2 2 2 e UO E E ETE E A A ii 22 LCS g aaa a a a a 22 AE E E a E A A 23 39250 RI 25 3 1 1 0 MO 0R KK 27 35152 7 Z s 3 Jb COD BIET este it ette reti Certe emer te hee Lee rest cti 34 ESCAPE iran E E IC ved TELS 41 4 37 V AW EIC d S18 amp TEFHODSRTERSS eee 45 b ABE EFEDRIGE oom HERRERA EA ME REED EE 53 BiacoreT100 5 1 FIFO DIVI 54 I MERE en INE 3 NR ERRORES 57 5 122 Ue Jur UT se heck EMT cocum SN 66 5 1 3 87 5 2
263. IIZO Cc cc 285 AFL cd RO A ENERO TR ROREM 286 8 EROR ounce up III MIND C LIN EID LI ID E UE LONE 289 8 1 kk 289 B2 NERO ECL Ejus uL i i i A fd 289 B s q9 dp pU TORIS e ertet oett eu ce Entf E Le LN LA 290 9 No 292 gt 0 AS OD EBBI out uice mi mutexs cigare El a 292 9 2 kkk 293 0 3 ao A i ID EEN AAE ANA A EE EEA 295 9 3 1 a aa ed AN DSE a O 295 9 3 2 ae 297 9 3 3 Leuten 298 9 3 4 kk 299 gos oce Er e Ec 300 S mcn Mi cde ordei im 4 0p I icr a D OM RR TEE 301 CE ME pP EEE NE ANAE A EAE E AA EA EEA EE AE EE AEAEE AE 303 9 5 es 305 9 6 aite A 0D 2 e 307 Biacore T100 1 1 1 1 1 1 1 1 gt gt gt gt Windows biacore
264. Lopes J 2 Variable Settings Commands Report Points Verification Y i Settings for Sample 1 Tupe Single cycle kinetics Method Variables Evaluation Variables Sample solution le variable Set property as variable SeupRun Sample 1 Sample solution ESOS Regeneration 1 Concentrations per cycle 5 Mj C Contact time s Contact time 120 s C Dissociation time s C Flow rate pl min Dissociation ime 500 s Flow rate 30 ulmin Flow path Both v N Estra wash after injection with abilization period Sample Type Single cycle kinetics Concentrations per cycle 5 contact time PX24 ANDR s 2 Dissociation time s 2 10 Flow rate ul min 30 l min Flow path Both Multi Extra wash after injection with Stabilization period
265. Method Builder Main Startup Beneral Settings Startup LMW kinetics 3 times as entered A Assay Steps i 2 Sample Sample LMW kinetics 1 time as entered Cycle Types Solvent correction Variable Settings t Solvent correction Solvent correction 1 time as entered Before after every 20 cycles Verification Control sample t Control sample LMW kinetics 1 time as entered Before after every 10 cycles Cycle Run List Assay step properties Base settings Recurrence Name Startup Repeat assay step within Purpose Startup b Connect to LMW kinetics v tribute 1 cycle type Assay step preparations 5 Number of replicates Temperature 3 times Buffer s entered 1 2 3 1 2 3 Q Order 1 1 22 3 3 CO Random Assay steps 5 D Assay steps New Purpose Cycle type 6 3
266. NT CONNEC UON cedet i 197 216 228 229 254 256 257 261 264 265 SPEC CONIC time arg TOW Fate o abet a a RO RO RARI AM GO CAN NS WR MRNA MUN MURUS 55 OD Eene oN NO E E E UT 59 98 177 198 216 260 ONG ENO SS 26 119 SANA OWN e Yn E 289 SO 59 96 98 102 125 150 153 176 197 216 232 247 254 255 256 257 Sues A aN TTE TE MEME MEUM A 90 STOD RU 65 108 133 164 182 202 222 245 273 SUFIGCE Info donre uo MN i 52 Surtdce Prepal GO senate ARRA DARREN MR ERROR RUE RBRUM BERTA ARA 28 354 41 visant aro NET i 286 T Mee w S MMTMIMRTMITTE 42 lemMOEeO e i 4 10 29 37 61 105 208 218 241 Jic edere i A 82 Biacore T100 QNSE giro PTT ERIT TRITT 82 TWO SINERGIO sena t 72 115 MO 98 99 153 154 U Uscaverdgecalbrotion UE VO saniser A 136 Uwe a i i 75 76 118 119 DO REA TEE M T 75 118 V ME 194 WeNIIGOOIN NN 100 156 249 266 MIQIMWWeIIOOSIIORKRKRRN000000000000000000000000OQ KN NM 18 49 W Wash Bilter DUBIO desi pteistuidetm RA 283 M BSHSDIU EPIO NR i oii 42 239 RD oi AU a 17 REA A iE E E EOS 253 254 255 257 258 264 265 Tim eT LM E E AEE EAE A CET 23 45 Uoc M e e LI ME EE uM MM E ME EE M M Ul 54 55 56 93 RI a E EE E T Gi 25 P PR NE cc oo 23 25 eo 22 o PAP E 14 SN 290 A zb mA e rm 305 306 IN 10 m m 53 2
267. Properties Chip id Chip lot no First use date 0s0507 1029 12114 B 7 2008 IFC type IFC105 Ez 12008 antibody CBia UsersiTi00ManualcsK immobilization of antibody blr Close Close id id Biacore T100 8 1 1 7 Series S Sensor Chip CM5 3 BR 1006 68 Series S Sensor Chip CM5 Certified 3 BR 1005 30 Series S Sensor Chip CM4 Certified 3 BR 1005 34 Series S Sensor Chip CM3 Certified 3 BR 1005 36 Series S Sensor Chip C1 Certified 3 BR 1005 35 DNA Series S Sensor Chip SA Certified 3 BR 1005 31 JRIKE9 7 Series S Sensor Chip HPA 3 BR 1005 33 Series S Sensor Chip L1 Certified 3 BR 1005 38 His tag
268. R C 22 V O ZRVI m a i T Sensor Chip T d ai CM5 W un Biacore T100 292 9 9 Biqcore T100 Evaluation Software
269. Reset All Filters i 0 0 Add Keyword Warfarin 1 Warfarin i Remove Keyword WWarfarin f hoe OEE NN Warfarin Warfarin Warfarin Warfarin Warfarin 1 MM centration Unit Naproxen Naproxen i Naproxen Naproxen iNapoxen Naproxen a Naproxen Naproxen Naproxen Furosemide Furosemide Furosemide Biacore T100 5 135 5 35 E Menubar Toolbar m Report Point Table Evaluation Explorer Work area Menubor Toolbar Evaluation Explorer W nsorgrom ot AbsResp Baseline 73 50 RelResp Bqseline RelResp Binding level Evaluation Explorer Baseline Binding level Binding stability Binding to reference Work area Biacore T100 136 5 Toolbar QD n Concentration Analysis A Using calibratio
270. Response Kinetics Affinity Fit Kinetics Create Curve Fc 4 3 Ligand antibody Sample antigen Temperature 25 C Add Fit Current Fits Model 1 1 Binding e EBEN Description zu c B t G4 Ch oc Quality Control Report Residuals Parameters Curve ka 1 Ms kd 1 5 Lnnr M Flow ul min kt RLI Ms Chi RU un BRE l225ET56 0 003754 3 065E 9 4 415E 11 1 063E 9 30 00 372ET12 0 2948 125E 9 0 2917 4 250E 9 0 4467 B 500E 9 2712 1 7 0E 8 0 4539 Biacore T100 118 5 Report k 1 Ms k 1 s FE SEE TERES XE EN Ko M FERE XE Rmax RU PX boss RI RU bulk effect Chi RU U value U Finish Evaluation Explorer 7 Sensorgram sensorgrams 3 Flot a Baseline Sample a Binding level Binding stability a Binding ta reference 7 Report Paint T able Hrepen E aunt T able Kinetics Affinity ma antigen
271. Sample 1 Dilution Startup Dumm y Startup Dummy a Startup Dummy 4 Calibration Biotin 100 5 Calibration Biotin 40 amp Calibration Biotin 16 Calibration Biotin 6 4 8 Calibration Biotin 2 56 a Calibration Biotin 1 02 a0 Contral Sample Control 1 33 3 11 Control Sample Control 2 11 1 12 Sample 501 2436 01 1 13 Sample 5 1 2436 01 1 Next gt Biacore T100 130 5 Te Concentration Analysis Rack Positions mes HORS O Loe dE Wall Microplate 00000 0000 eo0o0000 ALLALI LIO LLALL LIO 00000 Cceoc0cs00 Content 113 501 2436 01 113 501 2438 01 113 505 0001 02 113 ODIN 025 01 Empty Mix min capacity 114l Empty Mix min capacity 114l Empty Mix min capacity 114l Empty Mix min capacity 114l Empty Mix min capacity 114l Empty Mix min capacity 114l Empty i Mix min capacity 114l Empty Mix min capacity 114l 113 501 2436 01 113 501 2438 n1 113 505 n 01 02 113 ODIN 025 01 Empty Mix min capacity 114l Empty Mix min capacity 114l Empty Mix min capacity 114l Empty Mix min capacity 114l Sample Sample Sample Sample Calibration Mix Calibration Mix Calibration Mix Calibration Mix Calibration Mix Calibration Mix Calibration Mix Calibration Mix Sample Sample Sample Sample Calibration Mix Calibration Mix Calibration Mix Calibration Mix Sample 1 Conc ng n
272. Subtracted Sensorgrams C Zoom lock Remove Selection Biacore T100 114 5 ES Kinetics Affinity Fit Kinetics Create Curve Fc 4 3 Ligand antibody Sample antigen Temperature 25 C Add Fit Madek 1 1 Binding a Model x 1 1 Binding Add Fit Modek 1 1 Binding Binding Bivalent Analyte Heterogeneous Analyte Heterogeneous Ligand wn State Reaction Fit Biacore T100 5 115 5 25 B A 1 1 Binding A B AB 1 Bivalent Analyte A B AB AB B AB2 2 2 AB B 2
273. T100 5 3 Biacore A B A ie 5 123 B y RU ss A t RUvsC RE A A Slope SO 100 150 200 250 Time A Slope vs C B A A
274. U DIL M I a Ma UE M E 505 Biacore T100 ExXtEadwwasmerter ection AVR adu esdulleco tele enis Cerise NR 98 197 216 260 E ED 310116 1i RIALP AENT AAPS CMM CMM C PUE DUC ME DC UCM D PME UMANE LUCUS 229 F Edad IE LM LE D LI I d I I II 59 40 PRUNO RNC MMTT E 156 OWD OG hine A prt 15 28 47 58 98 99 101 125 155 154 157 175 196 215 239 240 259 267 gio Ero ERE OEE 15 29 47 59 98 99 126 153 154 177 197 198 216 239 240 259 GMO 126 260 G GENET aea E oi 94 148 235 249 250 251 261 262 General SENO Sorrente aa a tton d ten a a ed b i A 94 148 249 H HeterogerneousAPdlVEeicida tetendit teer estende iia ii tuspeiegiedieb usines tested eben 72 115 Heterogeneous ddd eA 72 115 HION VISCOSItY SOUTO SE 59 126 177 198 260 Ne m HQ 261 Jentenroje l 2o ife 88 A COUINO NT 26 27 28 52 53 mimobilizeation RESU ct P 43 ae SEENT A M AEE EEE E E EE EEE ET 140 Inoue CYC anina E tenta udebesive dtd ude n 159 JateiSiorete e ION Or 259 ect comndhd sorana T 18 19 48 49 ETSA EIE ES MR ESSE 261 K NEN O AOE A 24 54 55 73 75 77 85 93 116 117 119 193 194 205 208 EATA A ATERT A A TE T A TE A T IR 24 54 55 73 75 77 85 93 116 117 119 193 194 205 208 Eae E E A N AN AAN AT 24 45
275. ace Biacore T100 174 5 5 5 5 5 1 Toolbar Run Wizard Menu bar Run gt Wizard Assay Kinetics Affinity Binding Analysis Concentration Analysis Thermodynamics Assay Binding Analysis New Methods and Templates 2 Open Browse Open 6 T Binding Analysis Injection Sequence Detection Chip Capture GAMPLE 1 Sample Enhancement Regeneration REGENER
276. ack Positions Reagent Rack 2 CHR sr 9 OO 00000000 OQO0O00000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 Menu 73 5 Automatic Positioning T Automatic Positioning ISo 148 Negative control 148 Negative control 148 Negative control 148 Negative control 148 Negative control 148 Negative control 148 Positive control 148 Positive control 148 Positive control 148 Positive control 148 Positive control 148 Positive control 148 Analyte A 148 Analyte A 148 Analyte A 148 Analyte A 148 Analyte A 148 Analyte A 148 Analyte A 148 Analyte A 148 Analyte A 148 Analyte A 148 Analyte A 148 Analyte A 148 Analyte A 148 Analyte A bbm Change the order in which the samples are positioned by ordering the regions The first region in the list is positioned first Region Color Orientation Anchor Rack Vial Si Control sample Control sample Control sample Control sample Control sample Control sample Control sample Control sample Control sample Control sample Control sample Control sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample a 5 R1A12 12 Sample 1 Back Next gt Glose First Sort By
277. ase settings Recurrence Name Blank Repeat assay step within EE Sample O Every cycle wae on cycle type v Distribute n a occurrences evenly Run assay step once first Run assay step once last Assay step preparations Number of replicates Temperature 1 times Buffer As entered 1 2 3 1 2 3 Order 1 1 2 2 3 3 Random D blank Recurrence Distribute occurrences evenly 1 Number of replicates times Cycle Types Biacore T100 5 153 T Method Builder Main Overview e lup Description of selected cycle type General Satsl cycle Lopes jC Variable Settings Commands Report Points Verification aplure Settings for Sample 1 Tupe High performance Method Variables Evaluation Variables Sample solution buffer property as variable Sample solution Contact time 6 s Contact time s REA SO Dissociation time s Dissociation time 5 O ex 5 s C Flow rate pl min Flow rate 30 pl min Flow path Both v of m
278. bound Kinetics Affinity Select Curves Create Select evaluation mode Single mode 3 Batch mode Curves Cume Fe24 3 car w Ligand HZA wt Conc Flow dede cvet GS quim 46 30 47 30 0 0753 0 157 0 313 525 L4 v v v L4 L4 La L4 v Zoom lack Show concentration series Show blank s Show average blank s Select Evaluation mode 1 Single mode Batch mode Bath mode 5 14 Saqmple Show average blank s
279. cates Times 3 i Biacore T100 5 97 T Method Builder Main Startup General Settings Startup Startup 3 times as entered Es Copy EE S AssaySteps i a CE Sample Sample Sample 1 time as entered Cycle Types du MCN RM aa Move Up Variable Settings I Move Down Cycle Run List Assay step properties Base settings Recurrence Name Repeat assay step within Purpose Every Connect to Distribute cycle type occurences evenly Run assay step once first Run assay step once last Assay step preparations Number of replicates Temperature 1 times Buffer As entered 1 2 3 1 2 3 Order 1 1 2 2 3 3 O Random Sample Number of replicates Times Cycle Types Biacore T100 98 5 T Method Builder Main s gt Description of selected cycle type This cycle is used in sample steps and if used control sample steps mem Contains injection of sample and regeneration The sample is of the type single cycle kinetic with recommended 5 concentrations ie number of injections in each cycle Assay Steps Vuelo ois SN cycle
280. ct time z Elow rate ulmin Mim with Fraction of the mis solution Regeneration Solutior Reg solution High viscosity solution Contact time s Sample contact time Flow rate Mix with Regeneration Solution High viscosity solution contact time Flow rate Stabilization perion Next gt Biacore T100 Elow rate iumin Stabilization period NEN s s ul min Fraction Bj 75 25 75 40 s l min s 5 127 Te Concentration Analysis Calibration Curve Calibration Curve Anabte name Repeat calibration every sample cycle Calibration points Concentration Calibration Curve Analyte name Repeat calibration
281. ction Start Temperature kk Toolbar Thermodynamics 4 71 28 2 Biacore T100 5 209 E Thermodynamics Select Kinetic Data Create Import Kinetic and Affinity E valuations Ligand Sample antigen E v Model 1 1 kinetics steady state affinity Evaluation Temperature C amp Description ka 1 Ms kd 1 5 antigen 9 9 1 1 Binding 1 227E 6 5 571E 4 4 538E 10 i antigen 16 16 1 1 Binding 1105E 6 9 426E 4 8 534E 10 antigen 23 23 1 1 Binding 1 383E 6 2 038E 3 1 473E 3 antigen 3n 30 1 1 Binding 13801E 5 4558E 3 2 333E 3 i antigen 37 37 1 1 Binding 2 258BE 6 8544E 3 4270E 3 Import Thermodynamics Check All Next gt ES Thermodynamics Overview Create Thermodynamic Overview Temperature Descip on ka 17h kd 1 J 1 227E 6 antigen 16 15 1 105E 6 antigen 23 23 1 383E 6 antigen 30 30 1 501E 6 antigen 37 3r 2 250E 6 20 25 Temperature 15 z0 25 15 z0 25 30 35 Temp
282. e C3 Batch mode Curves Curve Ligand antibody v Sampe a Cycles Conc Flow Contact Time Diss Time nM ulmin s s yf 4 3 Ti Shown concentration series Show blanks Show average blank s Sqmple Show average blank s Include Biacore T100 112 5 ES Kinetics Affinity Select Curves Create Select evaluation made Single mode C3 Batch made Cures Cure Fe 4 3 v Ligand antibody v Sample Temperature 25 Cycles Conc Flow Contact Time Diss Time iii nM ul min s s 4 30 0 0 D gr 0 Zoom lack Show concentration s
283. e Fc 2 1 e Assay Step Purpose lt Overlay gt e Cycle lt Hyerlau gt i9 F gt Sensorgram 4 All sensorgrams RU Sensorgram E Plot 40000 C Zoom Lock Baseline Sample Binding level 35000 Binding stability Binding to reference Report Point T able 30000 Conditioning m Report Point Table Startup 1 25000 Startup 2 20000 Startup 3 Startup 4 15000 Startup 5 Thermo 1 10000 Thermo 2 5000 Thermo 3 Thermo 4 Thermo 5 HE 5 66 Keyword table Tools Keyword Table S Keyword Table LM Ex Warfarin iWarfarin Warfarin Warfarin Warfarin Warfarin WWarfarin tt MM Mofc Hn 4 Concentration Unit jWerfain DE 308 WM LOWE NEL Cosi Naproxen i Naproxen Naproxen ECT Naproxen Naproxen Hinn E MEM Naproxen i Naproxen Furosemide Furosemide Furosemide Biacore T100 204 5 5 67 iB Menubar Toolbar E Report Point Table Evaluation Explorer gt Work area Menubar Toolbar Evaluat
284. e RD xe ND yy Meqs Conc Meas Conc alibratianFreeLonc 1 5 _ 173 5 49 CFCA D ChiZ 5 SE standard error 10 SE 10 0 05 5 ug ml E 5 50 Evaluation Explorer 7 Kinetics Affinity ZEB u elobulin Backsp
285. elected Cycles Calibration Control Sample Id Cycles OK Custom Report Points Custom Report Paints Position Assay step purpose stabilityz n seconds after skop of Calibration injection Sample 1 Sample injection Control Sample Cqalibaration Settings Report Point lhrahinn Curve Settings Flow Cell Fc Report Paint baseline Use average calibration curve stabilit stability Biacore T100 5 143 5 4 CFCA Calibration Free Concentration Analysis CFCA Calibration Free Concentration Analysis
286. eni 1 1 11500 13 Sample antigenz 11500 Next gt Biacore T100 62 5 T Kinetics Affinity Rack Positions MI Conc nM MW Da 1 118 antigeni Sample 1 11500 118 antigeni Sample 118 antigeni Sample 118 antigeni Sample 118 antigeni Sample 118 antigeni Sample 118 antigeni Sample 118 antigeni Sample 118 antigen Sample 118 antigen2 Sample 118 antigen Sample 118 antigen Sample 118 antigen Sample 118 antigen Sample 118 antigen2 Sample 118 antigen Sample 118 antigen Sample 118 antigen3 Sample 118 antigen3 Sample 118 antigen3 Sample 118 antigen3 Sample 118 antigen3 Sample 118 antigen3 Sample 118 antigen3 Sample 118 antigen3 Sample 118 antigen3 Sample 334 buffer Startup 541 buffer Regeneration 1763 Gly HCl pH2 5 Regeneration D 5 3
287. eport Point Table C Zoom Lock Sample Startup Biacore T100 294 9 Menubar Toolbar E Report Point Table Evaluation Explorer Work area Menubar Toolbar Evaluation Explorer W nsorgrom piot Baseline Binding level Binding stability Binding to reference Work area Biacore T100 m Report Point T able Sensorgram window AbsResp Baseline RelResp Bqseline RelResp Binding level Evaluation Explorer 9 295 9 3 Evaluation Explorer Sensorgrum All sensorgrams amp Work area Sensorgram window 9 3 1 Sensorgram window
288. erature Temperature ga Biacore T100 210 5 Next gt Thermodynamics Results Create Calculated Thermodynamic Paramete vant Hoff Fitting function wan t Half Fitting function E vrina 21 5 22 j 28 3 3 258 3 3 3e 3 3 35e 3 3 4e 3 3 45e 3 3 5e 3 3 55e 3 3 6e 3 1T 14 Parameters Chi 0 9990 1A sk Eyring dissociation i F B 2 13 5 3j 28 3 3 258 3 3 3e 3 3 358 3 3 4e 3 3 45e 3 3 5e 3 3 558 3 3 6e 3 j 28 3 3 258 3 3 3e 3 3 35e 3 3 4e 3 3458 3 3 5e 3 3 55e 3 3 5e 3 1T 1 1T 14 Parameters Parameters Slope 1887 1 Ms Intercept 14 32 1 MsE R 20 7368 Slope 8785 1 s Intercept 17 87 1 sK FE 0 9907 lt Back 7 5 Rha Linear Non linear Fitting function van t Hoff Fitting function E pring Linear Farameter Mame Farameter Value AH kJ mol AS HKE mal TAS kJ mol Ala kJ mol ALp EJ K mal E 2 ong p eee eee AS t ass Hik mol 15 TAS ass kJ mol dz M Finish c 2Uw2d 2 Evaluation
289. eries Show blank s Shaw average blank s Next gt ES Kinetics Affinity Select Data Create Cumes Curve Fc 24 3 Ligand antibody Sample antigen Temperature 25 LC 30 Blank Subtracted Sensorgrams C Zoom lock z Biacore T100 5 113 0 5 24 Kinetics gt E 5 24 Remove Selection Blank
290. etics LMW kinetics LMW kinetics LMW kinetics Solvent correction LMW kinetics LMW kinetics s Close Cycles Assay step Startup 1 Sqample 7 Solvent correction 1 Control Sample 2 Enter the number of cycles you plan to run for each assay step in the left panel The list of cycles for the run is displayed in the right panel Assay Step Name Cycles Assay Step Startup 1 Sample 7 Solvent correction 1 Control sample Cycle Assay step name Assay step purpose Cycle type RICOINI Startup Startup Startup Solvent correction Control sample Control sample Sample Sample Sample Sample Sample Sample Sample Solvent correction Control sample Control sample Startup Startup Startup Solvent correction Control sample Control sample Sample Sample Sample Sample Sample Sample Sample Solvent correction Control sample Control sample LMW kinetics LMW kinetics LMW kinetics Solvent correction LMW kinetics LMW kinetics LMW kinetics LMW kinetics LMW kinetics LMW kinetics LMW kinetics LMW kinetics LMW kinetics Solvent correction LMW kinetics LMW kinetics v Close
291. ference a Garry over i Controls binding Evaluation Explorer D Plot Plot Work area Y 100XRU Da mro 8 ME lt Curve Mame Fcz 1 corr 4 Assay Step Purpose Over la 4 Gvcle number Overlay uM Tools v 1 0 RLIDa Zoom Lack MM adjusted respa binding 25 Cycle number 5 5 Biacore T100 232 5 5 82 Evaluation Explorer Plot Baseline Sqmple baseline
292. finity Create Curve Fc 4 3cor Ligand H A Sample Furosemide Temperature 25 C AddFit Model Steady State Affinity ww Concentration Cancel x M Y Req RU Model Steady State Affinity Fit Biacore T100 5 91 Kinetics Affinity Fit Affinity Create Lurve Fc 4 3cor Ligand H A Sample Furosemide Temperature 25 E Add Fit Current Fits Model Steady State Affinity Nm Steady State Affinity Description 55 5 Concentration Report Parameters ez 05 0 02560 Finish KD M Ko M Pr BEE AN Rmax RU offset RU bulk effect Chi RU Evaluation Explorer Toolbar j Kinetics Affinity
293. fter run C Specify analysis temperature after run m CD Data Collection rate 10Hz Detection 2 DualMuli Dual 2 1 4 3 Multi 2 1 4 3 2 1 5 1 4 1 S8 Sample compartment temperature 4 45 C 25C Concentration unit Buffer settings 8 After run mec BHSE SND Assay Steps Biacore T100 150 5 T Method Builder Main General Settings i5 Copy AssaySteps a Cycle Types Move Up Variable Settings Move Down Cycle Run List Assay step properties Base settings startup 3 times as entered m sample 1 time as entered blank Sample sample 1 time as entered Before every 10 cycles Recurrence Name satup Repeat assay step within Purpose Startup Connect to startup cycle type Assay step preparations Temperature Buffer CD Startup D Number of rep
294. g curent cycle please wait Abort cycle hn Ctri Break _ Ctrl Breakl _ Biacore T100 Evaluation Software Biacore T100 2 4 7 _ Menu bar Tools More Tools Maintenance Tools
295. gG x1 400 25 150000 Fc 4 3 1 HHuajml Protein pH5 O0 mause IgG x1 100 25 150000 mouse IgG x1 6400 Fc 4 3 Zoom lock 5 Flow Stplmin F Flow 1006 plimin Include view Sample mouse IgG 41 5400 Curve Fe 4 3 Show blank subtracted data Next gt Biacore T100 170 5 ES Calibration free concentration Select Data Create Settings 9 All sample series Single sample series Zoom lack Settings DO Single Sample series eje 3 3 M BUIDSBB C dS 732 o88B8l2 T
296. i ies eur mer un 41000 I EB 4006060 39000 a000 B Jy arag s6000 35000 34000 33000 32000 100 z200 300 II SH EO r eun II Time Z 60 bound 0 recovered 4 5 8 me ng 5 4098 053 060 4 33 4 94 0 08 Aids 23803 19427 Biacore T100 6 247 6
297. iation time FEE BST fes 120 s Extra wash after injection with Biacore T100 198 5 Regeneration Solution High viscosity solution 40 contact time 60s Flow rate 30 ul min Stabilization period Os Next gt Te Thermodynamics Samples Samples Sample id 0 000 HH 0 01251 0 02513 0 05015 a 1003 2006 0 01251 Run order 9 Aszentered Increasing concentration Sample id MW Da ai nM ug ml Concentration i E E ii B AJJf amp Next gt Biacore T100 5 199 5 62 Excel 2
298. igen Sample 11800 OOo Q Q e Q Q f 118 antigen Sample n 118nn 118 antigen Sample T 11800 i 118 antigen Sample 1 06 11800 o O e C C 118 antigen Sample 2 13 11800 118 antigen Sample 4 25 11800 v u Biacore T100 200 5 5 63 Menu Export Positions Menu gt Simple Position Import 5 64
299. in Dissociation time s Extra wash after injection with Stabilization period NN s Sample contact time 120s Flow rate 30 ul min Dissociation time fest Der fes 120s Extra wash after injection with Stabilization period Os Next gt Biacore T100 5 217 Te Kinetics Affinity Samples Samples Sample id SulFanilamide SulFanilamide 5ulFanillamide SulFanilamide SulFanilamide SulFanilamide 5ulFanillamide SulFanilamide SulFanilamide 5ulFanillamide SulFanilamide Azasulf amide AzosulFamide AzosulFamide AzosulFamide Azasulf amide AzosulFamide AzosulFamide Azasulf amide AzosulFamide Hun order 9 Asentered O Increasing concentration Sample id MW Do Concentration nM ug ml
300. inding stability Binding to reference Beta2 micro Chi RU 0 136664 md 9 307 9 6 File Save AS Save As My Recent D acuments G Desktop Mn Documents 5 Mn Computer File name Einetics amp ffinitu 1 1 interaction bme bul My Network Save as type Biacore T100 Evaluation Files bme wv Save in C Bia Users File name Save fig 9 2 Biacore T100 Control ia Biacore T100 Evaluation Biacore T100 S A AdcdkRepoOPbDOIFIU detained temen inde eiit A nidis etd nude N E ERA 21 Ada SaNentcornectUoN NO 226 AdidstroelE ror CoDtFolS scudo n Ou WR RR RO WR EO RR ORA ED ROME AN HOR EVER ONOEEOEG 187 NM 95 149 236 252 AIMTORIMMOD NZ WM 35 42 Analysis temperaturen Ri 10 29 37 61 103 129 178 197 218 241 252 ADDIICOBOR WIZGUCIS A i i i 14 ASSOL Step DPReDOFCOUOEIS NS hi 254 265 pscq oss EEEE N E EE nes Li uU
301. ing 8400 Controls stability Report Point Table 8200 mm Report Point Table 8000 Conditioning Control Sample 7800 Sample Solvent correction 7600 Startup 7400 7200 7000 6800 0 400 500 900 Time 5 17 Keyword table Tools Keyword Table Keyword Table Cycle Assay step purpose Sample Conc nM Mw Da TE Conditioning Startup buffer Startup _ buffer Add Keyword Startup buffer Sample antigenl Rename Keyword Sample antigen s Sample antigen Sample antigen i Sample antigen 1 n Sample antigen 11 E Sample antigenl 1 2 Sample antigen 13 Sample antigen2 14 Sample antigen2 i M 1 5 Sample antigen2 16 Sample antigen2 1 7 Sample antigen2 18 Sample antigen2 1 amp Sample antigen2 20 Sample antigen3 21 E Sample antigen3 22 Sample antigen3 23 Sample antigen3 24 Sample antigen3 25 Sample antigen3 26 Sample antigen3 27 Sample antigen3 28 Sample antigen3 v iT 2 3 5 e s Concentration Unit Biacore T100 88 5 Toolbar pA Kinetics Affinity 4 21 y pg A Surface
302. ints aplure v High performance v Method Variables Evaluation Variables p Set property as variable Sample 1 A Sample solution Regeneration 1 Contact time 36 s C Contact time s AR Dissociation time s Dissociation time 5 a 2 s Flow rate pl min ample solution Is variable Flow rate Is variable Flow path Boh v Predip Mi witi Frai Es wash after injection with Stabilizatiomgariod D Cycle types currently in Method Sample 2 Commands Sample1 Settings for Sample1 Type High performance contact time s 36 WEE Dissociation time fretis s 5 Flow rate ls variable Setup Run Variables Flow path Both Commands Regeneration1 Regeneration solution contact time s Flow rate umin 30 l min Flow path Both Variable Settings Biacore T100
303. ion Explorer W nsorgrom piot Baseline Binding level Binding stability Binding to reference Work area Biacore T100 m Report Point T able Sensorgram window AbsResp Baseline 73 50 RelResp Bqseline RelResp Binding level Evaluation Explorer 5 205 Kp kk 5 1 Toolbar A Kinetics Affinity ES Kinetics Affinity Select Curves Create Select evaluation made Single made C3 Batch mode Curves Contact Time Diss Time s s 1 06 30 2 13 30 4 25 30 30 30 30 30 30 La La L L L4 La La v L Shaw concentration series Shaw blank s Show average blank z
304. is solution Stabilizatior period after mix o s Estra wash after injection with C Stabilization period lp s D Startup 2 Commands Sample1 Settings for Sample1 Buffer Type High performance contact time s 36 WEE Dissociation time REUS s 5 Flow rate umin 30ul min Flow path Both Sample solution Buffer 27 7Z7f EGBDR Commands Regeneration1 Regeneration solution contact time s Flow rate umin 30 l min Flow path Both Biacore T100 154 5 T Method Builder Main Overview Cycle types Description of selected cycle type Genes sena ED Assay Steps Cdelwes 7 Verification Commands Report Po
305. itioning Control sample Solvent correction x j Rename ME Commands Report Points bul C arry oaver control Solvent correction Inject amp ndR ecover General IF then Commands Solvent correction Insert nse Solvent correction Assay steps New New D Biacore T100 t ii E q Fai Delete Copy Startup 5 Startup Thermo 5 4f Move Up sample Move Down t Is Cycle Run List Assay step properties Base settings M ame zzau ste Connect to Mot Connected M cycle type Assay step 1 6 265 Control sample 4 Control sample 1 time as entered Every 15 cycles A Control sample Thermodynamics 3 times as entered A Thermodynamics 1 time as entered Control ample 5 Control sample Control sample 1 time as entered Every 15 cycles A Mot connected 1 time as entered Recurrence
306. jecting a regeneration solution This solution is the typically the same as the solution used to recover the material captured on the sensor surface Typical recovery solutions that are MS compatible contain mild organic acids 0 2 12 v v trifluoro acetic acid TFA formic acid or acetic acid Settingvfor Regeneration 1 Ryfgeneration solution 0 52 TFA Contact time s Flow rate pl min Flow path Predip High viscosity solution Estra wash after injection with NI Stabilization period Method Variables Set property as variable Regeneration solution Contact time s C Flow rate pl min J Regeneration solution contact time Flow rate Flow path Setup Run s ul min 1 2 3 4 Te Method Builder Detection Detection Elow path Flow path Next gt Biacore T100 Bak mew Gee 1 2 3 4 5 241 Te Method Builder Cycle run list Assay step name Surface conditioning Surface conditioning Surface conditioning Inject and Recover
307. kBBROS2JZZmnetiue Start Save as Methods and Templates 7 Bia Users Don t Save Save Results From Run s Save in e T1 O0manual manual blr My Recent Documents My Computer e File name pHscouting o 3 My Network Save as type Result file blr Save in File nime Sqve Biacore T100 5x F Curve Sensorgram Fc 4 Sample C Lock scale kt iD gt m D EE t E E 150 250 300 Fc Time Window AbsResp SD LASD Slope RelResp Baseline Id Keywords in cycle 2 Value 4 18 0 5 35931 8 013 003 007 0 0 Yes baseline AssayStep Sample 4 83 0 5 358807 107 52 088 5747 958 9 No binding AssayStepPurpose Sample Buffer Buffer A CycleType pH Scouting Sample 1 Buffer name 10 mM Acetate 5 Sample 1 Ligand Sample 1 Sample Protein amp 10ug ml Temp 25 Online COMI Temperature 25 00 C Running immobilization pH scouting Sample c
308. l Protein pH5 0 mouse IgG x1 6400 6400 4 434E 10 2 838bE 06 0 377 25 150000 5 826E 11 Fc 4 3 100ug ml Protein pH5 0 mouse IgG x1 1600 1600 1 714E 09 2 142E 06 0 425 25 150000 5 826E 11 HITO BEDS S Biacore T100 172 5 libhration free concentration Evaluation Result Create Expand all cycles Dilution Meas Conc Factor M Fc 4 3 Fc 4 3 Fc 4 3 1Hunjml Prntein amp pH5 1Hiuniml Prntein pH5 U mouse IgG x1 6400 mouse IgG x1 1800 mouse Iga x1 400 6400 1600 400 4 434E 10 1 714E 09 100ugjml Protein A pH5 0 mouse IgG x11400 Fc 4 3 Meas Conc M Calc Conc M QC ratio QC EE Chi RU SE Meas Conc 5 49 Finish Biacore T100 7 D15E 09 Evaluation Explorer Concentration Analysis Calc Conc M 2 838E U6 2 142E Ub 2 806E 06 Qc Temp ratio ms s 0 377 150000 5 826E 11 0 425 25 150000 5 826E 11 0 391 25 150000 5 B26E 11 Zoom lack 12 Flow Srulimini Fitted 12 Flow Tlmini 13 Flow 1 0Cul min Fitted 13 Flow 1O0O pilimin t amp i amp Fable es 9 7 7 JL Di
309. l Settings X Delete Conditioning Conditioning 3 times as entered Copy AssauSteps A Inject and Recover T 5ample Inject and Recover 20 times as entered Variable Settings 3 Move Up M 9 Q Move Down Cycle Run List Assay step properties Base settings Recurrence Name nject and Recover Repeat assay step within Purpose Every cycle Connect to Inject and Recover v Distribute occurrences evenly cycle type Run assay step once first Run assay step once last Assay step preparations Gr of replicates Temperature 1l times Buffer As entered 1 2 3 1 2 3 Order 1 1 2 2 3 3 O Random Number of replicates Times Cycle Types Biacore T100 5 239 Inject and Recover T Method Builder Main vete Description of selected cycle type Inject and Recover The Inject and Recover cycle type regulates sample injection washing of the General Settings condmenmm kon system and recovery of the material that is captured on the sensor surface Within one cycle the sample injection and recovery se
310. licates times Biacore T100 Every cycle Distribute occurrences even ly Run assay step once first Run assay step once last 2 Number of replicates 3 times As entered 1 2 3 1 2 3 Q Order 1 1 2 2 3 3 Random 3 5 151 Ts Method Builder Main Startup startup 3 times as entered z n sample sample sample 1 time as entered LI blank Sample sample 1 time as entered Before every 10 cycles a T Save j Saves D Sample Number of replicates times Biacore T100 152 5 T Method Builder Main m Start up General Settings A Delete Startup Startup 3 times as entered N Assay Steps 2 CE Copy Sample 5ample Sample 1 time as entered Cycle Types Blank Move Up TAM Variable Settings Sample Sample 1 time as entered Before distribute 1 time Move Down Verification Cycle Fiun List Assay step properties B
311. ll Procedure Method Ligand Bound RU Final RU Target Reached 4 Target level Amine antibody 240 1 353 2 Yes Immobilization Results RU Target Reached Response Biacore T100 44 3 3 5 NHS Ta immobilization Results Chip Cf15 Response Response ERT Method inane Paundi Final BI Target Reached Target kvel Amine antiaodwy 10u0gml pH H Peiconcenitracion binding is boo Fart Preconcentration binding is too fast
312. ments ED Ti00 demo file Cj T1 00manual C Templates File name KineticsAffinity 1 1 interaction bme My Network Save as type Biacare T100 E valuation Files bme Biacore T100 5 245 Save in File naime Sqve Standby flow 5 84 Run Stop Run Biacore T100 AN This will stop the run stop Run Kun Stopped Finishing current cycle please wait Abort cycle hn Ctrl FH Break Ctrll Breakl Biacore T100 Evaluation Software Biacore T100 246 5 RU 341 K 42000 E r D EEUU nea qua
313. mplate Method Wizard Results Sensorgram Include event log for cycles Biacore T100 Current Cycle OBme C3 Al cycles Include event lag for cycles Method Current Cycle Range All cycles 3 23 90 CM5 N g
314. n F Calibration free Using calibration 2U v 2g 2 Concentration Analysis Create Calibration Control Samples Samples Calibration Curve Settings Flow Cell Fc 1 v Report Point stability Response Type Relative Response v Fitting Function 4 Parameter Use average calibration curve Calibration T able Calibration Curve Conc Response Calc Conc 9 Curve 1 we ng ml RU ng ml RU Biotin 1380 5 0 9948 1600 1400 1200 1000 800 600 400 Parameters for curve 1 200 Rhi 93 62 Hlo 1432 A1 21 42 A2 1 051 E 40 60 80 100 120 Chr 13 43 Concentration nami Concentration Analysis Create Calibration Calibration Curve Settings Flow cell BEANTICAIFHS t JL OR Report point Response type Relative Response Slope Fitting Function linear 4 parameter D Use average calibration Curve Biacore T100
315. nalyte A Sample A OQ e C C C e e C i 148 Analyte A Sample o Q s Q e s 148 Analyte A Sample 3 Q Q e Q O O O Q i 148 Analyte A Sample 7 Q Q Q O O O O 148 Analyte A Sample i 148 Analyte A Sample e e Q Q C Q Q 148 Analyte A Sample e o Q Q O Q 148 Analyte A Sample A Q Q Q e OO C O 148 Analyte A Sample i 148 Analyte A Sample 148 Analyte A Sample 148 Analyte A Sample 148 Analyte A Sample DELE a Back Next gt Menu 7j 5 Automatic Positioning amp 33 45v T Automatic Positioning Change the order in which the samples are positioned by ordering the regions The first region in the list is positioned first Region Color Orientation Anchor Rack Vial Si i First Sort By Control sample 7 Cyan Column Bottom left Sample Small ontent Ascending Sample E DarkBlue Column Bottom left Sample Startup I Crimson Column Bottom left Sample Wash E vellow Column Bottom left Reagent Small Tontent Ascending Small ontent Ascending Large ontent Ascending Small Content Ascending Y Y Y Solvent correction buffer 4 Ill Blue i Column Bottom left Reagent gt Biacore T100 5 181 Pooling Auto
316. nd Specify contact time and flow rate Contact time s Flow rate 10 mm C3 Blank immobilization Flow cell Method Amine Ligand NHS 10 umin 7 Aim for immobilized level Specify contact time and flow rate Blank Immobilization NHS Specify contact time and flow rate 420 s 10 umin Next gt 3 2 Specify contact time and flow rate 7
317. neticsAffinity multiple Rmax 37 1 1Binding 2 101E 5 1 692bE 4 8 053E 10 KineticsAFFinity KineticsAfFinity KineticsAfFinity KineticsAFFinity multiple Rmax multiple Rmax multiple Rmax multiple Rmax 892 30 90 2 91 89 90 90 91 37 1 1 Binding 37 1 1 Binding 37 1 1 Binding 37 1 1 Binding 1 742E 5 1 996E 5 1 675E 5 5 721E 4 1 809E 4 1 38E 9 1 779E 4 8 909E 10 1 867E 4 1 115E 9 gG 236E 4 1 440E 8 C pen empena rie Ads Append File File gt Append File Biacore T100 5 87 5 1 3 Evaluatton Biacore T100 Evaluation Software 2 CA drug blr ELE es Curve Name Fc 4 3 e JP ensorgram 4 All sensorgrams RU gy Plot 8800 C Zoom Lock Baseline Sample Binding level a 7 d 8600 Binding stability Binding to reference Controls bind
318. ngle Dual Mult 725 Single 1 2 3 4 Dual Las 38 Bs 455 Multi 1 2 5 4 2 1 4 3 2 1 5 1 4 1 Sample compartment temperature 4 45 C 15 10 DMSO Biacore T100 252 6 Vary with analysis temperature Analysis temperature Concentration unit 5 Buffer settings After run Biacore T100 6 253 Assay steps Te
319. nzert all four pump inlet tubes Place water nh the right hand tray and insert the water inlet tube A B C D Start l 4 5 Hesnrh and 5anitize Step 5 Leave tube in water Let tubes B C and D hang in the air A B C D Start hesnrh and 5anitize The Desorb and 5anitize procedure is completed Allow the zustem ta run in standby mode for at least 3 4 hours before performing a run 5 3 Standby flow 3 4 edo DLIA Prime 3 A HBS N HBS EP prime 1 A prime 1 Close
320. ody 1 ug ml nHH Sample antigen Temperature 25 E Add Fit Curent Fits Model 1 1 Binding 1 1 1 Binding Description Quality Control Report Residuals Parameters Curve ka 1 Ms kd C1 5 KD MY Rman RU Conc M tc Flow ul min kt RU Ms RI RU Chi RUP U L1531E 5 0 002610 1 705E 9 7 17BE 7 1 100E 9 2 231E 8 0 05105 2 200E 9 2 231E 8 0 2011 4 300E 9 2 231E 8 0 1965 Cycle 8 8 5 nM B 500E 9 1 0 1167 Cycle 9 17 nM 1 700E 8 2231E 5 1 62 ka 1 Ms k 1 s FERE TERES XE EN Ko M FERE AE AN Rmax RU RI RU bulk effect Chi RU U value U Finish Evaluation Explorer Biacore T100 76 5 7 Sensargram rJ All sensorgrams Plat gl Baseline Sample a Binding level al Binding stability a Binding ta reference l gt Report Point T able EE eunt T able Kinetics Affinity AJ antigen Toolbar zaKinetics fin
321. ompartment temperature current 26 C set 25 C Run time 9 min Estimated run time 18 min Immobilization pH Scouting Standby flow Biacore T100 Evaluation Software pH Control Software Biacore T100 Evaluation Software pHscouting blr A Thermodynamics BeK TE Curve Name Fc 4 e 4 Assay Step Purpose Sample e Cycle Overlay P ensorgram j Adjusted sensorgram RU Adjusted sensorgram F gt Report Point T able 3000 rj Report Point Table C Zoom Lock 10 mM Acetate 4 baseline 10 mM Acetate 4 5 10 mM Acetate 5 10 mM Acetate 5 5 Response 0 SD 100 Time 0 baseline pH Biacore T100 3 33 3 1 Immobilization pH Scouting Adjusted sensorgram Zoom Lack 10 mM Acetate 4 10 mM Acetate 4 5 10 mM Acetate 5 10 mh Acetate 5 5 H E D 3000 50 50 100 150 200 250 pH
322. on Anchor Rack Vial Si i First Sort By Control sample E Cyan Column Bottom left Sample Small i Ascending Sample E CarkElue Column Bottom left Sample Startup I Crimson Column Bottom left Sample Wash Yelaw Column Bottom left Reagent Small i Ascending Small Ascending Large Ascending Y Y Y Solvent correction buffer amp Il Blue Column Bottom left Reagent Small Content Ascending gt Pooling Auto Pooling_ Yes OK Automatic Positioning Biacore T100 6 273 6 7 7073 LORRIE Run Stop Run Hiarnre T100 AN This will stop the run stopRun Run Stopped Finishin
323. oned by ordering the regions The first region in the list is positioned first Region Color Orientation Anchor Rack Vial Si i First Sort By Control sample E Cyan Column Bottom left Sample Small i Ascending Sample E CarkElue Column Bottom left Sample Small i Ascending Startup I crimson Column Bottom left Sample Small Ascending Wash Yelaw Column Bottom left Reagent Large Content Ascending Solvent correction buffer amp Il Blue Column Bottom left Reagent Small J Content Ascending gt ol Auto Pooling_ Yes OK Automatic Positioning Biacore T100 244 5 Eject Rack Rack tray port OK Eject Rack Tray
324. oom lack Show concentration series Show blanks Show average blank s Multiple Amas Adjust Injection Start Next gt E Kinetics Affinity Select Data Create urves Lurve Fc 1 Ligand antibody 10ug ml pH5 Sample antigeni Temperature 25 C eee cre uh tmm r rn nM pl min s s 1 1 30 3 4 3 30 B 5 3 17 30 Blank Subtracted Sensorgrams E Zoom lock E Biacore T100 70 5 0 Kinetics gt 5 8
325. ore Methods Name Type Modified Ed Affinity in solution Method Builder 3 28 2008 Calibration Free Concentration Method Builder 3 28 2008 5 GST Kinetics Method Builder 3 28 2008 5 Inject and recover Method Builder 3 28 2008 5 Kinetics heterogeneous analyte Method Builder 3 28 2008 5 L1 liposome capture Method Builder 3 28 2008 D LMWw kinetics Method Builder 3 28 2008 5 LM screen Method Builder 3 28 2008 E NT kinetics Method Builder 3 28 2008 I Single cycle kinetics Method Builder 3 28 2008 Single cycle Kinetics Open Method Builder Main Overview 6 J Ki hi General Settings Biacore T100 5 95 T Method Builder Main Overview qe a General Settings Data collection rate Detection Sample compartment temperature Hz C Vary with analysis temperature Assay Steps Cycle Types u D Miscellaneous 5 Buffer settings Concentration unit M vi Postion 0 Name 0 0 0 O TTEN After run Specify analysis temperature after run D Data Collection rate 10Hz 2 Detection
326. ple antigen 20 Sample antigen3 21 E Sample antigen3 22 Sample antigen3 23 Sample antigen3 24 Sample antigen3 25 Sample antigen3 26 Sample antigen3 27 Sample antigen3 28 Sample antigen3 Ce J Cems ea Concentration Unit Biacore T100 110 5 5 23 Menubor Toolbar Evaluation Explorer Work area Menubar Toolbar Evaluation Explorer W nsorgrom Plot Baseline Binding level Binding stability m Report Point Table Binding to reference Work area Biacore T100 Sensorgram window AbsResp Baseline 73 50 RelResp Bqseline RelResp Binding level Evaluation Explorer 5 111 Toolbar p Kinetics Affinity 4 51 y pg A Surface bound Kinetics Affinity Select Curves Create Select evaluation mode Single mod
327. procedure The surface on ether sensor chips may be damaged by the solutions used Do not run this procedure below 20 C Do not abort this procedure after it is started Next gt hesnrh and 5anitize Required solutione fram Maintenance Ft Bl amp desarb solution 1 about 40 ml Bl amp desarb solution 2 about 40 ml Bl amp disinfectant solution about 80 ml Next gt Biacore T100 7 279 hesnrh and 5anitize Step 1 Flace 25 ml Bl desarb Solution 1 an the left hand tray and insert all Faur pump inlet tubes Place 15 ml Bl desarb Solution 1 an the right hand tray and insert the water inlet tube BIAdesorb Solution 1 25 ml 15 ml 2 A B C D BlAdesorb Solution 1 1 25 mD BIAdesorb Solution 1 15 ml Start 1 2 liEsnrh and Sanitize Step Wipe the pump inlet tubes with a moist tissue Place 25 ml Bl desarb Solution 2 an the left hand tray and insert all Faur pump inlet tubes Flace 15 ml
328. quence can be Assay Steps repeated up to 14 times The total sample volume maximum roughly S0 microliter will be equally divided over the number of repetitions Note that salts and detergents present in recovery deposition wash Cycle Types Ji 2 solutions and running buffer can reduce sensitivity in mass spectrometric MS Overview analyses on the recovered sample Variable Settings Commands Report Points 4 Capture v Sample solution Method Variables Set property as variable Tonic ume s C Sample solution Inject amp ndR ecover 1 5 ulmin C Contact time s Flow rate C Flow rate pl min C wash solution C Recovery solution Wash solution 5 C Deposition solution C Deposition solution volume Flow path Recovery solution Incubation time s Deposition solution B mM NH4HCO3 Deposition solution volume ul Number of repetitions 5 Sample solution contact time s WR ul min Flow path 1 2 3 4 BENI Wash solution Recovery solution
329. re Maintenance Kit type 2 BlAdesorb solution 1 0 5 96 SDS BIAdesorb solution 2 50 mM Gly NaOH pH 9 5 Maintenance Tools Wash Buffer Tubing Start Wash Buffer Tubing Select tubes ta wash Next gt Wash Buffer Tubing This procedure removes adsorbed material from the Flow system The procedure is divided into three steps Total run time is about 30 minutes HOTE Use the Maintenance Chip for this procedure The surface on other sensar chips may be damaged by the solutionis used Do nat run this procedure below 20 C Do not abort this procedure after it is started Next gt Biacore T100 284 7 Wash Buffer Tubing Step 1 Place 20 ml Bl amp desorb Solution 1 on the left hand tray and insert tube BIAdesorb Solution 1 20 m Stqart 1 2 Wash Buffer Tubing Step 2 Wipe the tube with a moist tissue Place 20 ml Bl desarb Solution 2 an the left hand tray and inzert the tube sn
330. re T100 9 303 9 4 Sensorgram window Caption Undo Cut Scale Copy Granh Export Curves taridlines Legend AZ Jvo 558 Scale Scale Y Scale Auta Auto Lagarithmic Lagarithmic Auto Autn px Ty2 J4L e Min Max A Scale Y Scale Auto amp uto Lagarithmic Lagarithmic OK Biacore T100 304 9 Legend Legend Position C2 Hidden o Q Top 5 Right C2 Bottom Right Hidden OK Gridlines Gridlines m AMS Major Gridlines E Minor Gridlines Y Axis Major Gridlines Minor Gridlines Mojor Gridlines
331. re T100 Evaluation Software Eject Rack Rack Illumination Insert Chip Set Temperature Preferences More Taals et Temperature Analysis temperature Sample compartment temperature 4 45 COD88BH CagaxE LC OK 15C Status bar 4 D temperature 27 Z7 D FESIC Ell d S temperature 25C Biacore T100 1 11 1 2 4 Toolbar Eject Rack E
332. rotrein amp 20ug ml pH5 Method Amine Procedure TimeAndFlow Online COM1 Temperature 25 01 9C Sensor chip CMS Sample compartment temperature current 26 C set 25 C Running standby remaining time 4 0 days Response Fina RU Ce immobilization Results Chip CM5 Response Response Flow cell Procedure Method Ligand Bound RU Final RU 4 Time and Flow Amine Pratrein z ug ml nH5 1383 8 1513 9 Biacore T100 40 3 3 4 Response Bound Final 2 Bound Final NHS EDC Finol Bound NHS
333. rst value Low Second value High Medium Biacore T100 5 187 RL Binding level 7 Zoom Lock 30 4 25 20 d 15 Z 4 Startup Sample T value 10 Control sample a 10 t E values 2 TES atte m 5 5 5 10 15 20 25 30 35 40 45 Cycle number 5 58
334. s 2 Open Browse Open Biacore T100 Te immobilization Immobilization Setup Flow cell 1 ES Immobilize flow cell 1 Flow call 2 F E Immoabilize flow cell 2 Flow cell 3 Immabilize flow cell 3 Flow cell 4 F Imrmobilize flow cell 4 Chip type CM5 Flow cell 4 makie fisso ell d Method Aim for immobilized level Ligand Dilute ligand 3 Specify contact time and flow rate Target level RU wash solution 50 mh NaOH C3 Blank immobilization Flow cell Aim for immobilized level Method Amine Ligand Target level RU Wash solution 50 mM NaOH Ne
335. s entered Before after every Verification Control sample t Control sample LMW kinetics 1 time as entered Before after ewery Settings for cycle type LMW kinetics Sample 1 varies by cycle 60s 600s Carry over control 1 SetupRun Report points Assay steps General settings Expand lI Collapse All General settings Biacore T100 6 251 T Method Builder Main General Settings B Data collection rate Detection Sample compartment temperature Hz C Vary with analysis temperature Dual 4 T Variable Settings SPSOUE setinas Concentration unit Buffer settings yewcson M J PBS 5 DMSO After run Specify analysis temperature after run General settings 6 D Data Collection rate 1Hz 10Hz 10Hz 1Hz Detection 3 Si
336. sition 5KO AAM e contact time 60 120 Inject HL Cancel Eject rack tray ll Menu bar Commands Eject Rack OK Inject command s OK T Biacore T100 Control Software regeneration check blr ilz File Edit View Commands Run Tools Help Ex dZEJ 739 Cyde 1 x Curve Subtracted Fc 4 3 C Lock scale ding 1 stability 1 In R3 x Xb baseline 1 1500 50 100 150 200 300 350 Time Window AbsResp LASD Slope RelResp Baseline Id Keywords in cycle 1 Value 5 1881 4 0 04 0 01 0 0 Yes baseline_1 5 4238 7 018 1525 2257 2 Mo binding 1 5 4325 5 0 21 203 23451 No stability 1 Flow 30 Flow Path 3 4 Online COM1 Temperature 25 00 9C Running manual run Sample compartment temperature current 25 C set 25 C Run time 6 min
337. t HBE 5 52 Excel Excel Excel txt To Kinetics Affinity System Preparations Frime before run Normalize detector Temperature settings Analysis temperature C Sample compartment temperature C Prime Normalize Analysis temperature 25 0 Sample compartment temperature 25 C Biacore T100 Cycle Run List Te Binding Analysis Cycle run list Conditioning Startup Startup Startup Control Sample Sample Next gt T Binding Analysis Rack Positions Reagent Rack 2 v ere e O 5 dB well Microplate OO ee e e e eo QOO M w A Ch c CO w o oO OS OS Se OS OOo oeeo oeeo OT TI J oOeeeo OT TI J eee IIl 00 00 buffer buffer buffer Cycle Assay step name Sample 1 Solution Control O O c A Roc
338. tion time s 1 Flow rate ul min 30 l min Flow path Both Multi Commands Regeneration Remove Variable Settings Biacore T100 100 5 T Method Builder Main Assay steps Define variable handling for each amp ssay Step General Settings Define all values at run time Assay Steps Define all values in method Define some values in method and otbefs at run time Cycle Types Variable Settings 73 oe Sample Solution SetupRun t 77 V TOZDAN EDA CEZADA iBln EO 3 Define all values at run time Define all values in Method JFRiBIE E CAJZJd lEFKUIS XV v KECY hE LTE
339. trol sample Control sample QeoOooOO0000 00000000 00000000 00000000 00000000 00000000 Oe000000 O0000000 148 Analyte A 148 Analyte A 148 Analyte A 148 Analyte A 148 Analyte A 148 Analyte A 148 Analyte A 148 Analyte A 148 Analyte A 148 Analyte A 148 Analyte A 148 Analyte A 148 Analyte A 148 Analyte A Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample eMe Back Next gt Menu 7j 5 Automatic Positioning amp 338 5v Te Automatic Positioning Change the order in which the samples are positioned by ordering the regions The first region in the list is positioned first Anchor Rack Vial Si E Column Bottom left Sample Small Column Bottom left Sample Small Column Bottom left Sample Small E Column Bottom left Reagent Large 1 Bottom left Reagent Small Region Color Orientation Control sample E Cyan Sample E CarkElue Startup I Crimson Wash Yelaw Solvent correction buffer amp Il Blue First Sort By Ascending Ascending Ascending Content Ascending J Content Ascending Column gt ol Auto Pooling_
340. uired olution fram Maintenance Kat Bl amp normalizing solutian Next gt Start Hormalize Normalizing please wait Time left 00 08 16 Normalize The Normalize procedure i completed Close Standby flow Biacore T100 286 7 Desorb and Sanitize E 1 eb Kg x M d rJ Biacore Maintenance Kit type 2 BIAtest solution HBS N Buffer 150 ml 10X Buffer Series S Sensor Chip CM5 BIAtest solution 1 5 ml CM5 Dock HBS N EDR C Prime Menu bar Tools More Tools
341. up I 4 45 52 027 P 45 46 50 52 ctn IUBE E E RT TN 46 233 RE a a A EE A A A AANA AAAA AAAA AAT 24 75 77 91 187 xc ecc UM LEM LE PL LL LA I M E E 74 76 117 118 Biacore T100 wn AURI NER 63 105 131 161 180 200 220 243 272 Sp 10 2 62 104 130 160 180 200 219 270 61 102 129 138 144 145 178 185 199 218 268 FAZIE ROS P pem 66 87 109 134 165 168 171 183 203 224 L A PIO TE ee NE ED 285 AATA T T EEEE ENO SENET SOET SENEESE 281 286 287 IATL P i 239 277 JA o RE 2 143 IIIF II E a Nt 45 53 54 93 gd DIUS T I C L m 24 45 53 174 211 230 231 ML MI UA M M M 289 LIC Mic 73 116 DU cns M M MILL M MEUM ML LM Ind UU 73 116 ch 5 P T 194 210 CH 2 028 BS MIR cetestistete tos a bsec i 70 113 2 0 EE 292 295 Ed Rau RAO ASA e T 7 mm 5 lu rS A ID 290 uzbeh4zii RI ec ONERE D 0 0 fii 45 BEVERTE is oti oaa bua etr uta rh bod 45 53 58 59 93 193 197 211 213 216 233 261 BS i e i 123 126 260 TIAE zo 37 EOD BIER oieri c i 139 227 5 EE n MM MM M M MM UL UE 123 BEDS Bf Bis nier e ERR EE EOE 53 197 211 220 261 ELI E
342. ution Froteirs Tag Contact time s Flow rate Ll min Surface regeneration This surface wash will be run ance at the end of each cycle Solution Bm NaOH Ligand Solution contact time s 60 s Flow rate ul min 10ul min Surface regeneration Solution 50 mM NaOH Next gt te Immobilization pH Scouting System Preparations Frime before run Normalize detector Temperature settings Analysis temperature C Sample compartment temperature C Prime Normalize 4525 Temperature settings Analysis temperature 25 C Sample compartment temperature 25 C Next gt Biacore T100 AEN 38 ProteinA 10ug ml Sample 10 mM Acetate 5 5 38 iPrntein 10udg ml Sample 10 mM Acetate 5 38 Protein 10ug rml Sample 10 mM Acetate 4 5 38 Protein 10ug ml Sample 10 mM Acetate 4 198 50mM NaOH Regeneration U
343. ution F3 E Te E RS BT CD e H contact time s Number of injections Startup Solution Number of cycles 3 Solvent correction 5 7 Number of injections Repeat after Sample cycles Temperatures Analysis temperatures 25C 5 Sample compartment temperatures Next gt Te Thermodynamics Injection Parameters Sample Contact time 120 s Elewrate 30 l min Dissociation time s C Extra wash after injection with Regeneration Soho IlyHClpH25 High viscosity solution Contact time s Flow rate ulmin Stabilization period d s Sample contact time 4 OW 120s Flow rate E 30 ul min Dissoc
344. xt gt l 37 40 Biacore T100 3 43 Te Biacore T100 Control Software immobilization of antibody blr mf ilc File Edit View Run Tools Help o a c m 2 E lc EN Cycle 1 Curve Sensorgram Fc 4 antibody Lock scale D 4 500 1000 2000 m o Time Window amp bsResp SD LASD Slope RelResp Baseline Id I Keywords in cycle 1 Value 37 0 5 354575 27 25 3 21 14 43 HNA PreConc1 Chip CM5 43 0 35567 1 32 30 0 62 17 58 HNA PreConc2 Ligand antibody 57 0 35828 7 35 23 028 1883 HNA PreConc3 Method Amine 87 0 353833 33 31 053 17 81 HNA PreConc4 Procedure TargetL evel 132 0 371328 29 70 0 28 15 88 Na PreConc5 TargetLevel 250 531 0 3668384 0 10 0 02 0 05 0 0 Baseline 1 1114 0 367378 317 0 80 1 65 33 3 EDC NHS 1185 0 36808 4 0 06 0 06 0 02 110 0 Fulse1 1258 0 368821 0 21 0 02 011 183 7 Pulse2 1332 0 36977 0 0 23 0 04 012 278 5 Pulse3 1382 0 363330 X 0 20 0 06 010 300 6 Pulse4 Online COM1 Temperature 25 00 9C Sensor chip CMS 4 4 4 4 4 4 4 4 4 4 4 Cn cn cn cn an an a ai nn Sample compartment temperature current 25 C set 25 C Running standby remaining time 4 0 days LS immobilization Results Chip CM5 Response Response Flow ce
345. ysis temperature 25 C Sample compartment temperature PC Prime Normalize Temperature settings Analysis temperature 25 C Sample compartment temperature 25 C Cycle Run List amp 2 UU v 2d c ma Startup buffer 2 Startup buffer 3 Startup buffer 4 Solvent correction 5 Control Sample CESA 50 2 Sample SulFanilamide Sample SulFanilamide 8 Sample SulFanilamide 0 0075 9 o Sample SulFanilamide 0 157 a0 Sample SulFanilamide 0 3131 11 Sample SulFanilamide 0 625 a2 Sample SulFanilamide 1 25 13 Sample SulFanilamide 2 5 a4 Sample SulFanilamide 5 15 Sample SulFanilamide 1 15 Sample SulFanilamide 0 625 Biacore T100 5 219 Next es Kineticsffinity Rack Positions L E Reagent Rack 2 ka 3 eo 148 SulFanilamide Sample e C X 148 5ulfanilamide Sample 148 SulFanilamide Sample Qe 148 Sulfanilamide Sample e C XC 148 Sulfanilamide Sample e 148 Warfarine Sample 1 148 Warfarine Sample NU 148 warfarine Sample B c h E F G 148i Warfarine Sample dE Well Microplate 148 Warfarine Sample 148 Warfarine Sample 148 Warfari Sampl O0O00040010 D uen en y C3 C 148 Warfarine Sample

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