Biacore 2000 （Ver.3） 取扱説明書
Contents
1. pU ROS G Rmax Da RU Da s s 50 000 Do 1 000 RU 1 20 000 Rmax Rmax 20 000 x 1 000 50 000 x 1 40080 Biacore Rmax 100RU2. O pH Sensor Chip CM5 me MEME bound final lj aa __
3. HBS HBS EP 10 mM HEPES 0 15 M NaCM3 mM EDTA 0 005 96 Surfactant P 20 pH7 4 HBS P 10 mM HEPES 0 15 M NaCl 0 005 96 Surfactant P 20 pH7 4 HBS N 10 mM HEPES 0 15 M NaCl pH7 4 0 22um CM5 Biacore 2000 Instrument Handbook 1 7 1 2 3 Command gt Rack Base Rack Base THERMO amp amp WE2UvVZUGIRS S
4. 0 5mM 20mM CHAPS 40mM Octylglucoside 0 1mg ml BSA 50mM NaOH Zea New Application Wizard OUrface Preparation 5 55 Binding Analysis Kinetic Analysis Customized Application Create Mew Template Cancel qi The Surface preparation application wizard guides you through the essential steps in preparing the sengor surface for analysis including immobilization pH scouting ligand immobilization regeneration scouting and surface performance test Open Template C N Program Files BIACORE 2000 Guide Methods N Wizard Template HPA Chip Coupling blw Biacore 2000 Instrument Handbook 172 9 Application Wizard Ta Chip Goupling blw Wizard Template Wizard Customized Application Notebook Chip Coupling Injection of liposome after surface wash Stabilization af the lipid layer with high Flow rate arid 0 injection Check degree ol coverage af the sufrace with Save As
5. Bulk Effect 44 5056154 foc vC S79 23 ng ml ug ml Kp M 80 1 10 10 6 Oug ml 1 2 n 2 6 0 2 n 2 CL SEBA 6122 770 1 KERHA c Sec G Sc 2 8 Regeneration O
6. L BlAevaluation 1 1 Langmuir with mass transfer r Biacore 2000 Instrument Hondbook 9 Application Wizard 133 9 6 2 2 Linked Reaction 1 1 wizard We ia s n 1 1 Of 7224 400 Sd BIAevqluation Run Run Application Wizard Kinetic Analysis v T Kinetics Analysis Steps k E Kinetic Analysis Steps Concentration Series Control E periments Analyses the dissociation phase
7. Biacore 2000 Instrument Handbook 10 1 1 2 5 SPR _ Normalize BlAmaintenance kit BIAnormalize solution 0 5 ml R2F2 Tools Working Tools Normalize Working Tools X Cancel Normalizes the signal in all flaw cells sg this procedure when the apto interface is changed b when the temperature is changed c when a new sensar chip is dacked and maximum sensitivity is required Total run time is about 5 minutes Last Run Time 12 May 99 15 30 Start 7 5 Normalize SPR CD 2 Biacore 2000 Instrument Handbook 11 aa 2 ac L C Sucrose
8. im Biacore 2000 Instrument Handbook 140 9 Application Wizard 9 7 2 1 1 mouse lgG 1 R2A1 INJECT 40ul2 10mM Gly HCl pH2 20ul 1 R2F3 mouse IgG 10 baseline mouse 196 20 bound baseline 60 regeneration baseline Run Run Application Wizard Zea Mew Application Wizard Surface Preparation Binding Analysis kinetic Analysis Customized A
9. TIE Dee Biacore 2000 Instrument Handbook 1 9 1 2 4 Command gt Set Temperature Da Set Temperature x cet l emperature OK Cancel Temperature 4 40 CO8BB OK 4 4 40 states window Temperature 777 UZ F ESOD mee d 2 qr Ic 5 2289 5056 186562 9 5
10. a5 2 B S B 2 Fisch CRUS VN i x iB OANA HE S
11. pH 1 2 pH pH pH 5 50 ug ml 10 mM pH6 100 10 mM pH 5 100 10 mM 100 Precnncentration of mouse IgG pH5 J m e e Response
12. 50mM NaOH 30 Application Wizard 86 Biacore 2000 Instrument Handbook 3 21 5 50 ug ml 10 mM pH 6 100 10 mM pH 5 100 10 mM 100 p m WI eges i Run Run Sensorgram dA gt Er Detection made subtraction c Fc 1 c E Fe fme x Comes x CES Ej Fc OK 5 9 Flow Set Flow g F ndo ul min Cancel 10 umin OK Command gt Inject D
13. 1 1 1 1 binding Biacore 2000 Instrument Handbook 138 9 Applicqtion Wizard 9 7 Customized Application M wizard Run Run Application Wizard a Mew Application Wizard Surface Preparation Binding Analysis kinetic Analysis INTE temized Amnlicatian Create New Template qi Cancel This wizard allows you to program and run customized applications For purposes that are not covered by the application specific wizards Customer Application Creat New I
14. Step Next gt Biacore 2000 Instrument Handbook 9 Applicotion Wizard 119 a Binding Analysis Regeneration 10 mM Glycine pH 1 9 i 4 Binding Analysis Rack Positions THERMO E REAG A THERMO U serine OO OO 5000000000 Biacore 2000 Instrument Handbook 120 9 Wizard 742 gt i Binding Prepare Run Px THERMO right hand rack position Position Volume Content B aample 51 AZ Bl aample S2 A3 B aample S3 AJ 60 sample 54 B sample B sample A B aample S7 AB B aample S8 AB 60 sample 59 A10 B aample 510 H1 B aample 11 B2 B aample S12 B3 B aample 813 Bd 60 sample 514 B5 B aample 515 B6 B aample S15 B aample S17 ED Sample 518 Prime Before Run ly Standby Flow After Fun Help Menu lt Bark Start Close
15. rine _ S E vm i IC 525 576 D DR gt On gt m EILAN ORAAL 91 5 3U 977 0u 3 JALAN 177510 Ab IC 60
16. AL 9 00 17 30 www gelifesciences co Jp e maill Web 2009 GE GE UKAS T 169 0073 3 25 1 ISO 9001 2000 TE
17. Sanitize 3 1 MilliQezK do U Mad 2 7 7 78134 C Prime 1 2 Biacore 2000 Instrument Handbook 6 2 Amine Coupling HSA T ug ml Fa cn c c e n m 2 1200 1400 1600 1800 Tools Working Tools Prime Flush Rinse 2 Tools gt Service Tools Unclogging IFC 56 lt 6 3 i2
18. OK For Report Point bound IF RelResp In Flow Cell 2 1 Value 50 Comparison In less Than THEN Exit Cycle Biacore 2000 Instrument Handbook 9 Application Wizard 157 4 DETECTION 2 1 FLOW 20 B vsz E Flow Path LOK Cancel Help Er Flow Path OK 2 DETECTION 2 1 F FLOW 20 FLOWPATH 1 J 2 DETECTION 2 1 F FLOW 20 EF FLDWPATH 1 INJECT Sample 50 INJECT END Biacore 2000 Instrument Handbook 158 9 Application Wizard ul min
19. Serial amine coupling DEFINE APROG immob CAPTION amine coupling Fc 1 2 3 4 FLOW 20 DILUTE R2B1 R2B2 R2B3 50 INJECT RPOINT INJECT INJECT INJECT RPOINT DETECTION APROG APPEND R2B3 140 baseline b R2A1 140 R2B4 140 R2F3 20 immob 1 2 5 4 immob standby Instrument Handbook 76 8 2 NHS e Uo 0 19 7 SS s 0 01 DEFINE APROG immob CAPTION amine coupling FLOW 10 FLOWPATH 1 2 DILUTE R2B1 R2B2 R2B3 50 INJECT R2B3 70 RPOINT baseline b FLOWPATH 1 INJECT R2A1 70 FLOWPATH 2 INJECT R2A2 70 FLOWPATH 1 2 INJECT R2B4 70 INJECT R2F3 20 RPOINT immob DETECTION 1 2 APROG immob APPEND standby NHS NHS
20. ZLI EJb1c 5 CO Ca Detection 1 2 JI 1 2 3 4 Detection Biacore 2000 Instrument Handbook 44 4 13 1 View Plot Single 1 A cu H OPa cU Is View gt Plot Overlay 9 dor SO View Plot Curve Classes CurveseoEIRg SC 2 20D 2 0 S
21. Cea Binding Results 1st Resp 1st Resp2 Base Shift Elow Cell Injections Cycles E Resp 1st Sample Hesp 2 H ss ENEN Baseline Shift Relative Group bu Cycles Baseline Level ET PPPPPPLPPPLE LLPLLPLLPLPPLPPPLPLL Resp1 Resp2 FB Biacore 2000 Instrument Handbook 122 9 Application Wizard 9 6 Kinetic analysis 9 6 1 Concentration series kK wizard Run Run Application Wizard Run Application Wizard Surface Preparation Binding Analysis Start EEEE Use Template Customized Application Cancel qii The Kinetic Analysis Application Wizard guides you through the steps in determining kinetic parameters for an interaction helps vou to design and perform crucial control experiments and gives immediate Feedback in the form af apparent rate constants based on 1 1 interaction modell Kinetic Analysis
22. NHS CON COO COO CONHCH CH OH 176 166 Biacore 2000 Instrument Handbook 18 3 2 SA 70 0005 SA
23. CES SEX C S rebinding CO wizard 23010189 7 1 1 Laongmuir with mass transfer Run gt Run Application Wizard Kinetic Analysis T Kinetics Analysis Steps x Kinetic Analysis Steps Concentration Series Analyses the binding of one analyte concentration at a range of How rates Results are presented as an averlay plat aligned to the time of injection Help
24. i 1blw Sequence _ EE TEMP 25 DETECTION 2 1 Flow 20 p BASELINE baseline 5 KINJECT Sample D 40 120 KINJECT END 20 RESPONSE bound 5 INJECT TOmM HE 20 e INJECT END amp 0 RESPONSE regeneration 5 Next gt Biacore 2000 Instrument Handbook 9 Application Wizard 153 Henu lt Back gt Close Sample 10 5 Repl Run Oeder 118 Zea ll Cycles Hun Drder IsEneed 1 E M Use Separate Vials for Different Solution Variables Repi Igi 25ug ml Igi 12 5ug ml Igi 5 25ug ml 1 Ig 3 125ug ml 1 gG 1 S625uq mi Menu M lt Back gt Cloze Next gt Biacore 2000 Instrument Handbook 154 9 Application Wizard s S20 THmh iGhy HCI pH2 sample ID IgG 25udg ml Rg AJ Ji Sample ID IqG 12 5ud ml Rg Sample ID lqG B z25ug ml Rz 90 Sample ID IgG 3 125udg ml Sample ID IqG 1 5525ug ml THERMO THERMO LJ E K E KK LJ E K QOO 0006 QG9OUQu sure that the c
25. ORU Baseline RelResp 00187 0 0 Biacore 2000 Instrument Handbook 2 15 Injection of several concentration of Sucrose 20 40 60 Fc Time AbsResp Slope Baseline RelResp 19 1 855 124959 0 06 Yes 0 baseline i RelResp i RU Injection of several concentration of Sucrose 40000 35000 30000 25000 Response 20000 15000 10000 5000 0 100 200 300 400 500 600 Tirne 700 800 T Time bsResp Slope Baseline RelResp Id 1 5 124959 0 baseline 1 127 5 154223 0 02 No 2927 0 2 sucrose 1 2985 183097 0 08 No 5813 7 4 sucrose 1 4725 240101 0 24 No 11514 1 8 sucrose 1 6485 353049 0 92 No 22808 9 16 sucrose Biacore 2000 Instrument Handbook 16 2 2 3 File
26. D sia c S 42 CD D pH 3 4 30 1 Biacore 2000 Instrument Handbook 38 4 4 1 l ra Run gt sensorgram D ga de zi Detection OK 43 210 kaka 20 50ul min i
27. Biacore 2000 Instrument Handbook 9 Application Wizard 165 9 8 Wizard Template Biacore 2000 Wizard Template C Program Files BIACORE2000 Guide Methods Wizard Templates Z Concentration Analvsis blw FA Chip Coupling blu 7 Ligand Thiol Coupling blw 7 Manual Ex1 Inhibition assay blu 2 Manual Ex2 Consecutive Regenerationblu 2 Manual Ex3 Positive Control b lw 7 Manual Ex4 Positive Control every fifth cycle blw FA Chip Coupling blu G Chip Preparing blw FA a Chip Preparing blu FA surface Thiol Coupling blw bro eT l Z y HPA eUT 0 voc Mec 5 NTA NTA SA Template d
28. 151 50mM 30 ul Biacore 2000 Instrument Handbook 74 8 MAIN DETECTION 1 1 APROG immob immob APPEND Standby Standby 5 ul min 51 APPEND Standby Immobilization Fc APRD0G Time AbsHesp Slope Baselime RelResp z immob 312 5 119560 002 Yes 0 Baseline 2 mmob 2123 5 151202 0 23 Ho 3164 2 immob Biacore 2000 Instrument Handbook 8 75 72 1 2 3 4 4 20 ul min
29. Stqndby 96 Standby 80 ml 5 MiliQO Biacore 50 Sa s o a Tels a e C Prime 242 2 MiiQO Tools gt Working Tools Prime Close 16mm MiliQ 3 ml AN R2F3 C 5 Tools Working Tools Close Biacore 2000 Instrument Handbook 52 5 Re 2 5 2 Undock Command Undock Undock Sensorchip S Bicare A Fr Ay IVE a Ea 5 3 O Dry
30. 6 21 NHS pH8 5 pH 10 mM pH 8 5 100 ug ml Biacore 2000 Instrument Handbook 20 3 3 1 CM CM
31. wizard D Run gt Run Application Wizard gt Surface Preparation Stqrt TOG ourface Freparation Surface Freparation Steps Immobilization pH Scouting Immobilization Regeneration Scouting Surface Performance Test Biacore 2000 Instrument Hondbook 106 9 Application Wizard 2 5mim Q0 20ul min 900 Next gt Surface Preparation Hack Positions Next gt i Biacore 2000 Instrument Handbook 9 Application Wizard 107 T Surface Preparation Prepare Run left hand rack position Position Volume pl Content 1 To DSA Prime Before Run Iw Standby Flow After Run Hep Menu lt Back Start Close
32. Biacore 2000 Instrument Handbook 50 4 18 View Method ER Edit View eem TT Tools NIS He E Scale J Adjust Scale c E Cycle 1 Curve Sensorgram RU BE hkb A 1 ug iml tn immobilized Human Serum Albumin 14500 Beference Line F8 14100 Baseline pg 13700 Plat Single 13300 Plot Overlay 12900 bethad 12500 Mntehnnk 12100 Markerz 11700 Extended Event Log 11300 10900 10500 0 Keywords Statys 1 1 11s En File Edit Command IE Bun Tools Window WS m x F l oir e icl E cscie 1 Curve senscrgsamFenn FR 286 nm Agfialyte 20000 19500 19000 19500 18000 17500 17000 16500 16000 15500 15000 100 200 400 500 DD Tl ann ann 1000 Time Fc APROG Time amp bsHesp Slope Bazel HelHesp
33. MAIN DEFINE APROG CN 9 es DEFINE APROG injection FLOW 5 INJECT R2A1 5 END MAIN DETECTION 1 APROG injection END MAIN APROG injection DEFINE APROG injection DEFINE APROG MAIN 77 0 DEFINE APROG MAIN APROG m m E m 2 Biacore 2000 Instrument Handbook 6 8 APROG MAIN APROG injection APROG 2 DEFINE APROG injection FLOW INJECT END MAIN DETECTION APROG Injection APROG Injection APROG injection DEFINE APROG injection FLOW INJECT DETECTION APROG Injection DETECTION 2 APROG Injection DETECTION 3 APROG Injection DETECTION 4 APROG Injection Biacore 2000 Instrument Handbook 8 65
34. 50 mi 5 SI AN CES D EUER DNA D Wet m eka AY1F 2525 50 ml 4 1595 00 Ss Dock
35. Biacore 2000 Instrument Hondbook 6 53 6 6 1 Biacore Working Tools Tools Working Tools Prime Dock TIFC 18739 MilliQe 6 30 8 A lt ERA 9 520561 Prime 2 53 2 EIN CUIUS Flush 3 Rinse 7 1 ORDY 7027 DID CRo 2 8 2000 Instrument Hondbook 54 6 Desorb 1 IFC 5 SE BIAdesorb solution 1 0 5 505 BIAdesorb solution 2 50 mM 9 5
36. 50mM NaOH Next gt Biacore 2000 Instrument Handbook 96 9 Application Wizard NHS RU NHS wizard 50mM NaOH NHS 21 2 1 3 Blonk
37. Add 558 Surface Preparation Ligand Settings Ligand Settings M ame Ligand Injection Time 2 Flow Rate 10 ulmin Run Settings Flowcell Help lt Back Hent gt Close 237 P uMmin Next gt Biacore 2000 Instrument Handbook 88 9 Application Wizard 78 ourface Preparation Wash Next gt Single Injection 20 60ul min 50mM NaOH 30sec 1 CIUS 2 Wash Method Two Injections pH Biacore 2000 Instrument Handbook 9 Application Wizard 89 60 Ligand in 10 mM Acetate pH 5 50 60 Ligand in 10 mM Acetate pH 5 00 60 Ligand in 10 mM Acetate pH 4 50 60 Ligand in 10 mM Acetate p
38. 20 kb 4 76 kb 1 20 kb 1 5 90 kb 5 5 150 kb Biacore 2000 Instrument Handbook 8 61 8 Biacore 1 MAIN 1 DEFINE APROG MAIN DEFINE APROG END 1 MAIN 1 1 MAIN MAIN MAIN MAIN
39. D Pp s s 21654 2 Antigen 1 25 pg ml Antigen 1 12 5 pg ml Antigen 2 25 pg ml Antigen 2 12 5 ug ml DEFINE APROG assay PARAM pos id conc CAPTION Binding of id 96conc to immobilized antibody FLOW 20 KINJECT pos 60 180 0 10 RPOINT baseline b 5 20 RPOINT bound i INJECT ReF5 20 2 00 RPOINT regeneration END MAIN DETECTION 2 1 APROG R2A1 antigen 1 25ug ml APROG R2A2 antigen 1 12 5ug ml APROG R2A3 antigen 2 25ug ml APROG R2A4 antigen 2 12 5ug ml APPEND standby Biacore 2000 Instrument Handbook 8 83 id conc CAPTION Binding of 96 id 96conc to immobilized antibody id conc AT 1 0 5l c RII Binding of Antigen_1 25 ug ml to immobilized antibody Cuts 2 o0 ms Binding of Antigen_1 12 5 u
40. 16 t te e hh e m o e s s s s s s s s s s s s s s s s s s s tm n s n ng n ng 55153 e 5 20 86 44 88 47 28 3 30 61 26 Biacore 2000 Instrument Handbook s m Lp ee GR 9 9 9 om om om om om om m om on n 19 5 5577270 s s s s s s s s s s s s s s s s m s m o s s s tm 0 0 n t9 32 39 p lic e 61 Z s s s s s o o s s s s o s o s o n 53 5 e 78 DDD ANT s s s s s s ls 19 2 TSS e s s s s s s s s s s s s s 17 165 176 s s s s s s s s 5 17 23 92 0 7 722 o s s s s s s s s o s s s s s s s s so s s s s s t t ng 9 9 9 n9 ng n 9 14 15 35 J gt 2 e s s s s s s s s s s o
41. pH pH5 Biacore 2000 Instrument Handbook 3 23 3 2 Application Wizard 92 61 3 2 1 l File gt Open amp 2 2795 BIACORE File Look in E Methods kinetic 3 L_thiol 2 Recover 2 S thiol E Fraction Gradient File name 5mine Files af type fan BIA Files b M Cancel C Program File BIACORE 2000 NGuide NMethods Amine Open f 1 Program blIm Result blr 3 BlAevaluation ble
42. 5 20 30 Biacore 2000 Instrument Handbook 6 8 RPOINT b 0 10 RPOINT 10sec b RU 0 RU ERV TVA OMBZDTBXMBS CU CE DEFINE APROG assay FLOW 5 INJECT R2A1 5 0 10 RPOINT 10sec b 0 20 RPOINT 20sec 0 50 RPOINT 30sec END n m E m 2 Time Window AbsHesp 5D Slope Baseline ld 3430b 5 108 27 10sec 34437 7 29 41 1435 131 1 2sec 3453 2 5 24 n 135 5 08 Biacore 2000 Instrument Handbook 8 69 4 4 2 DETECTION
43. DETECTION DETECTION DETECTION DETECTION DETECTION Biacore 2000 Instrument Handbook FLOWPATH FLOWPATH FLOWPATH FLOWPATH FLOWPATH 8 73 DEFINE APROG CAPTION Amine coupling FLOW 10 10 ul min DILUTE R2B1 R2B2 R2B3 50 NHS EDC R2B1 EDC R2B2 NHS R2B3 50 R2B1 50 200ul fE INJECT R2B3 70 R2B3 70 ul 10 ul min 7 43R30o E 70 30 ul INJECT R2A1 70 R2A1 70ul 70 30 ul INJECT R2B4 70 R2B4 70 009 5 70 ul 30 Hlo NHS INJECT R2F3 10
44. 10 000 15 000RU ka ka Kinetics Parameter RU 200 x 1 5 x 1000x 1 S x S 50 kDa 120 kDa S 1 200 1 1x 50 000 120 000 83RU 75 1000 1 1x 50 000 120 000 417 RU MANUAL INJECT 32
45. 5 Preparation Immobilization Aim for Immobilized Level 95 Specify Flow Rate and Injection Time 101 Next gt Biacore 2000 Instrument Handbook 9 Application Wizard 95 9 2 1 Aim for Immobilized Level 5ul min NHS 7 dei Surface Preparation Immobilization Procedure Immabilization Procedure C Specify Flow Rate and Injection Time Help lt Back gt Close Aim for Immobilized Level Next gt 252 Surface Preparation Aim for Immobilized Level Help lt Back gt Close RU
46. 1 296 Sucrose 2 496 Sucrose 8 896 Sucrose 4 1696 Sucrose 2 1 100u Ju LIT Run Run sensorgram Detection 1 100ul min OK Biacore 2000 Instrument Handbook 12 2 r m Command gt Inject i E Injection ner ei LN 10 ul Start Injection Injection of several concentration of Sucrose Response 296Sucrose i Biacore 2000 Instrument Handbook 2 13 RU Injection of several concentration of Sucrose 40000 35000 30000 25000 Response 20000 15000 10000 5000 0 100 200 300 400 500 600 700 800 Time Fc Time absResp Slope Baseline RelResp 19 1 85 5 12495 9 0 06 Yes 0 baseline 1 127 5 154229 0 02 No 2927 0 2X sucrose 1 298 5 18309 7 0 08 No 5813 7 4 sucrose 1 4725 24010 1 0 24 N
47. Biacore 2000 Instrument Handbook 8 Al DEFINE APROG FLOW FLOWPATH INJECT R2A1 FLOWPATH INJECT 2 1 FLOWPATH INJECT 2 1 FLOWPATH INJECT 2 1 DETECTION APROG FLOWPATH 1 R2A1 FLOWPATH 2 FLOWPATH 3 FLOWPATH 4 R2A1 Biacore 2000 Instrument Handbook 72 8 5 C NProgram Files BIACORE 2000 Guide Methods Amine bIm DEFINE APROG immob CAPTION Amine coupling FLOW DILUTE INJECT RPOINT INJECT INJECT INJECT RPOINT RACK RACK DETECTION APROG APPEND 10 R2B1 R2B2 R2B3 50 R2B3 70 baseline b R2A1 70 R2BA 70 R2F3 10 immob 1 thermo b RACK1 thermo b 2 thermo a immob standby MAIN RACK1 thermo b Command gt Rack Base DEFINE APROG
48. 120ul 30ul Cancel am Manual Injection Hode Injection 2 Start Volume t Manual Accumulated injected volume pl Remaining volume in pump 332 wl Manual Start Stop View Baseline 0 za CCE 200 vtm rtm anh agb Greig he De Die Ye Tek ib AERE RU 2000 Instrument Hondbook 36 3 UJ ROBEK NHS EDC INJECT NHS EDC A Time bsResp Slope Baselne RelResp Id Ligand ame 677 5 17 7060 001 Yes Baseline anti alb 160ugZml 3 Am4 1240 5 17823
49. 33 1 NHS NHS EDC 033 R c ZA Command Inject 72 v 29 5 Injection Start Injection 7 NHS 2000 Instrument Handbook 34 3 I UL EQUUM Ig Command gt Inject 278 Injection Injection in 1 INJECT RINJECT HHILRINJEDT COINJECT BIGINJECT Contact Time Consumption More gt gt MANUAL INJECT Start Injection Y x Injection in Fe 2 INJECT pr Manual Injection Mode FIT C Accumulated injected volume pl Remaining volume in pump Load Loop Biacore 2000 Instrument Handbook 3 35 5 3 Load Loop Load Loop Eram Position 2al wolume 50 ul Needle Position OK
50. Biacore 2000 Instrument Handbook 8 77 2 DEFINE APROG DEFINE APROG DEFINE APROG immob 1 CAPTION Amine coupling FLOWCELL 1 FLOW DILUTE INJECT RPOINT INJECT INJECT INJECT 2 00 END DEFINE APROG immob 2 10 R2B1 R2B2 R2B3 50 R2B3 70 baseline b R2A1 70 R2BA 70 R2F3 20 immob CAPTION Amine coupling FLOWCELL 2 FLOW DILUTE INJECT RPOINT INJECT INJECT INJECT RPOINT DETECTION APROG DETECTION APROG APPEND 10 R2B5 R2B6 R2B7 50 R2B7 70 baseline b R2A2 70 R2B8 70 R2F4 10 immob 1 2 immob1 2 immob2 standby Biacore 2000 Instrument Handbook 78 8 4 1 2 3 4 28925 INJECT 2 1 70 35 20 10 1 2 3 4 70 35u 201 10110015 Gradient surface Response DEF
51. 2 3 stoabilization Time 5 1 n 2 0 Enter Next gt Biacore 2000 Instrument Handbook 124 9 Application Wizard Next T x N For reliable kinetic analysis vau should run at least 5 different concentrations per analyte I nnre 5 Ignore BIAGORE N For reliable kinetic analysis v
52. PU mw INJECT pos PARAM MAIN APROG assay R2A1 R2A1 pos 2 R2A2 3 R2A3 esc oSv JC Sec ele APROG asay MAIN DEFINE APROG assay PARAM pos 0 10 2 20 2 00 END MAIN Biacore 2000 Instrument Handbook FLOW INJECT RPOINT RPOINT INJECT RPOINT DETECTION APROG APROG APROG APPEND 20 pos 40 baseline b bound R2F3 20 regeneration il assay R2A1 assay R2A 2 assay R2A3 standby 8 81 2
53. Stqndby Flow After Prime Before Start Save Wizard Results As Save n 3 Demo File name sco Save as Fiesults bir Cancel Sqve Biacore 2000 Instrument Handbook 108 9 Wizard Regeneration Scouting Response Analyte bound In last injection 31 0 AU Total amount analyte bound 30RU set tn 1004 Status Idle Regeneration Add Analyte Biacore 2000 Instrument Handbook 9 Application Wizard 109 2 Surface Preparation Regenerate Regeneration Solution 10 ncine 2 A 10 ml Glycine I TU Glycine Remove 50 mh Injection Settings Injection Time 0 5 Flow Rate 20 ulmin Position mA Required volume 65 pl The door is unlocked until you press Start Help S
54. 5 2 CM4 5 3 BIR CM5 4 C1 CM3 5 SA a E cDNA EIS NTA His Tag Fusion Protein
55. gu e tes File New Method DETECTION 1 Run gt Run Method status window DETECTION 1 5905 MAIN os Run Run Run Method DETECTION 1 DETECTION 2 DETECTION 3 DETECTION 4 DETECTION 1 gt 2 gt 3 gt 4 MAIN Biacore 2000 Instrument Handbook 6 8 MAIN Working Tools Run Prime 2 Rinse 2 3 Biacore MAIN 1
56. C Rabbit IgG 1 25ug ml 207 2 6560 5 Tools gt Service Tools Unclogging 2000 Instrument Hondbook 56 6 6 4 EACS S CENNI 5 CM5 2 HBS EP 9mm 2 BIAtest solution BIA maintenance kit Desorb Sanitize Dock HBS EP C Prime 2 Thermo_A BIA maintenance kit BIAtest solution 1 ml 9mm R2F1 9mm 4 R2E1 R2E4 Tools Testtools System check 40 llest Tools x System check raj System check evaluation Cancel Checks detection unit Flow cell Factors pump function autosampler performance and IFC sample loop and valves Tota
57. 2 2 R2A1 R2A2 R2A3 3 101 20 ul 30 ul EF DEFINE APROG assay PARAM 9ovol FLOW 20 i INJECT pos vol RPOINT baseline b RPOINT bound INJECT R2F5 20 RPOINT regeneration DETECTION 1 APROG assay R2A1 APROG assay R2A 2 APROG assay R2A3 APPEND standby pos vol 2 PARAM vol INJECT pos vol MAIN APROG assay pos vol 2 s 1 R2A1 10 il APROG assay R2A1 10 2 R2A2 20 il APROG assay R2A2 20 3 R2A3 30 ul APROG assay RaA3 30 APROG PRAM 2000 Instrument Hondbook 82 8
58. 2 CODO 1 10 ug ml 20 50 lt lt 9 IZEN AmE 150mM GE 1 1 2 pH 10mM AFRE 19 21 86 UST FARAO pH irc 20 Bc qap S s s EDCIN ethyl N 5 dimethylaminopropyl carbodiimide hydrochroride NHS N hydroxysuccinimide 1 M ethanolamine hydrochloride pH 8 5 22 0D 89 883 215 w BIOAXNetTvCcUssgS3slCttU EDC NHS 10 ml MilliQ
59. wizard 2 ec Wizard Template 165 Run gt Application Wizard Fd BIACORE 3000 Control Software on COMI Ele Edt View Command Tools Window Help D Hun sensorgram E d ia FAE 2 Hun Applicaton vvizardi Run Data Collection Rate Run Application Wizard Surface Preparation Binding Analysis Kinetic Analysis Customized Application Start Use Template Cancel qii The Surface preparation application wizard guides vou through the essential steps in preparing the sensor surface for analysis including immobilization pH scouting ligand immobilization regeneration scouting and surface performance test Surface Preparation 237 U Start 6 22 5779 5 Biacore 2000 Instrument Handbook 9 Application Wizard 93 78 ourface Preparation Surface Preparation Steps Immobilization Immobilization Regeneration Scouting Surface Performance Test Immobilization Next gt fd Surface Preparation Immobilization Aniecomim M CM5 Amine Coupling Next gt Biacore 2000 Instrument Hondbook 94 9 Application Wizard
60. Biacore 2000 Instrument Handbook 9 Application Wizard 151 2 7 D Wizard Protein 2 2 1 1 KINJECT 40ul 2 120 10mM Gly HCl pH 2 20ul 1 mouse IgG 10 baseline mouse 196 20 bound baseline 60 regeneration C baseline Biacore 2000 Instrument Handbook 152 9 Application Wizard 1 wizard Sample ID Predip Solutian Sanm Bu Injection Mode KINJECT Dissociation Time Sample Solution Vary by Cycle
61. 1 Concentration series ka M1s1 s1 wizard 2 Control experiments Mass transfer control wizard Linked reaction 1 1 wizard 2 Customized Application wizard Biacore 2000 Instrument Handbook 86 9 Application Wizard 9 1 Immobilization pH Scouting 10mM wizard pH 10mM Run Run Application Wizard Run Application Wizard Surface Preparation Binding Analysis Kinetic Analysis Customized Application Start Use Template Cancel qii The Surface preparation application wizard guides vou through the essential steps in preparing the sensor surface for analysis including immobilization pH scouting ligand immobilization regeneration scouting and
62. 1M pH 8 5 5mM 8 5mM Biacore 2000 Instrument Handbook 170 9 Application Wizard a Aldehyde Coupling blw Prepare Run THERMO A right hand rack position Position Volume pl Content A1 Full Ligand Buffer A To Ligand 1 140 CYanaborohydride E1 135 EDC E2 135 MHS E3 Empty vial for Mix minimum capacity 200 Lil E4 95 Ethanalamine F1 Full 5 mh Hycdrazine 95 5 mhi Hydrazine F3 165 Regeneration Solution Prime Before Run Standby Flow After Run _ Heb Menu lt Back Start Close Start Save 27 7 v2 Sc S887 pg Response Fc APROG Time AbsHesp Slope Baseline HmHgsn Id 4 immnh 305 5 110249 0 03 Yes 0 Baseline 4 immnh 2402 5 22870 0 0 16 11845 1 LIGAHD 4 immnh 42435 201216 0 09 9096 8 immob Biacore 2000 Instrument Handbook 9 Application Wizard 171 9 8 2 HPA
63. NHS Jb o 25 4 Surface Preparation Aim for Immobilized Level Flow Cell Ligand Name Wash Solution RU Eee ine EN lt Back Nest gt Close Biacore 2000 Instrument Handbook 9 Application Wizard 97 a Surface Preparation Rack Positions Poston ww cmew EDC Coupling Solution 115 NHS Caoupling Solution Empty vial tor mixing min capacity 200 pil 210 Antibody 5 Ethanolamine 50 50 NaOH THERMO_ L REAG THERMO 0 555500 3y000201 0000222200 O OJnl8 5980 CO Oo OO a Qe 0 0 0 0 O 600 O0 sure that the corect rack s are installed in the autosampler when you start the wizard _ Heb Menu lt Back Hest gt Clase Next gt Biacore 2000 Instrument Handbook 98 9 Application Wizard 2 Surface Preparation Prepare Run IIHERMO A right hand rack
64. gt Prerun Method 2000 Instrument Hondbook 28 3 3 2 4 Run Run Method Amineblm C Ne BIACURE Q Save changes in method Amine bln Yes No i Save Results Az Save n 3 Demo File name immobilization Save as type Fiesults bir Cancel Save Biacore 2000 Instrument Handbook 3 29 RU Amine coupling 50000 45000 40000 35000 amp 30000 25000 20000 15000 200 400 600 800 1000 1200 1400 1500 1800 Fc APROG Time AbsResp Slope 9 O Result View Method ag LT eL Scc CL
65. 14 Bulk Effect Bulk Effect Bulk Effect Biacore 2000 Instrument Handbook 4 45 15 28 r L lt la Command gt Stop Inject WASTE Injection Stopped Remaining 5 ample Dispense remaining sample to 16 EI do Zea Injection Sample la Volume 1 g ul Contact T Ime s Consumptior 40 Jl Mare gt gt CD More gt gt Recovery Recovery Fraction Collection Mone To Position rZal Recovery Volume 60 O Fractions Ex
66. v E Customized Sequence Ei ml slt Al DETECTION 2 1 9 KEYWORD 9 KEYWORD Conc F FLOW 10 INJECT 20 E 10 BASELINE baseline 5 d INJECT END E 20 RESPONSE bound 5 INJECT TOmM Gle HCI pHz 20 e INJECT END RESPONSE regenaration 5 Next gt 161 Biacore 2000 Instrument Handbook 162 9 Application Wizard Zea 1 Cycles Hun Drder As Entered Use Separate Vials for Different Solution Variables Standardi Slandard2 standarda Slandard4 Slandard5 Unknown Lnkhnwyhg Unknowns I I I Menu lt Back gt Llose Repl Analyte Conc Conc ug ml 1 100 1 2 unknown 1 2 Next gt Biacore 2000 Instrument Handbook 9 Application Wizard 163 7m 1 Rack Positions Unkno
67. 8 Bitmap Image My Computer 3 Briefcase Bia C v A Users Work Offline Close Demo Hosoya lwata san File New Folder Enter 9 Biacore 2000 Instrument Handbook 2 Explore Start Program Windows Explores i C Bia Users File New gt Folder Enter 19 Bia Users 20 CD 1
68. 3 RU 14 APRU G Tie window AbsHesp SD 5 Baseline HelHesp 14 kinetics 4 rH 50 23787 1 016 Yes baseline kinetics 4 335 0 20 2450 5 5 39 6 78 No 7135 bound DEFINE APROG injection FLOW 5 0 10 RPOINT 0 20 RPOINT 0 30 RPOINT END MAIN DETECTION 1 APROG Injection RPOINT Biacore9 2000 Instrument Handbook 6 8 0 20 20sec 20 Time Window A amp bsRHesp 50 Slope Baseline HelResp 108 27 37 57 1 sec 34437 7 2841 14 95 2 sec 3453 2 5 24 2 0 HN 7 3 sec
69. RPOINT 10sec 20sec 30sec FIAI FERA AbsResp RelResp Z B RU 5 Window 5 Biacore 2000 Instrument Handbook 8 67 DEFINE APROG assay FLOW 5 INJECT R2A1 5 RPOINT 10sec RPOINT 20sec RPOINT 30sec INJECT R2A1 RPOINT 10sec RPOINT 20sec RPOINT 30sec END MAIN DETECTION APROG END AA as 202 0 10 RPOINT 10sec INJECT 2 1 5 EI R2A1 10
70. TG T F lo uu k Ei Flow Rate ad ml min Var by Cycle Cancel Help Refill OK 2 DETECTION 2 1 FLOW 20 EF FLOWPATH 1 INJECT Sample 50 INJECT END Biacore 2000 Instrument Handbook 9 Application Wizard 159 5 Keyword Zea Keyword Cancel z Keyword Analyte Protein 2 2 1 1 20Vmin IgG 5 5 J R2A1 R2A2 R2A3 R2A4 R2A5
71. Customized Application 138 a E D E a AAA AATA AAE 139 NEEDLE PEDI PEE 140 9 8 Wizard Template lr 165 OPENED WI 166 9 82 Z TE eo ient a 171 ORE CE M v m 176 rojo E 181 Biacore 2000 Instrument Handbook 1 1 1 1 1 1 1 1 gt gt gt gt 30 Biacore 2 am mo ES dom uL MM l ER ZwcwZWmW Biacore 2000 Instrument Handbook 2 1 1 1 2
72. Start Ces Kinetics Wsie Steps Kinetic Analysis Steps Control Experiments Mass Transfer Performs analysis with a series of analyte concentrations to determine kinetic parameters Help 4 Back Nest gt Close Concentration Series Next gt Biacore 2000 Instrument Handbook 9 Application Wizard 123 Ta Kinetics Assay Principle Assay Pririciple Binding Using Capturing Molecule amp M ame Ligand Flow Rate 5 ymin Injection Time 3 min Stabilization Time mir lt Back gt Close Direct Binding Binding Using Capturing Molecule Direct Binding Next gt ferm BP Binding Using Capturing Kinetics Concentration Series E Use Flow Cells z Using 1 as reference Flow Rate 20 Stabilization Time 0 min Injection Time 2 min Dissociation Time 3 min Hun Order AsEneed Entered 150000 150000 150000 150000 lt Back gt Llnse 20ul min 2 3
73. 2 Baseline Level Response Level 3 Baseline Level Response Level Biacore 2000 Instrument Handbook 116 9 Application Wizard 9 5 Binding analysis wizord Run Application Wizard gi Hun Application Wizard Surface Preparation Binding Analysis Kinetic Analysis Customized Application Binding Analysis Start 2u Binding Analysis Assay Principle Direct Binding Next gt Binding Using Capturing Molecule Biacore 2000 Instrument Handbook 9 Application Wizard 117 252 Binding Analysis Injections Settings Use Elow Cel
74. 4 40 a Cycle Settings Temperature 25 CC by Cycle Cancel Help we Set Detection Detection Fci g HgFg Yan Cycle Selling the detection made will also set the initial Flow path OK egi emma t i gt TEMP 25 DETECTION 2 1 F FLOW 20 FLOW 20 0100 ul min V FowRae umin Var by Cycle Refill Cancel OK Biacore 2000 Instrument Handbook 9 Application Wizard 143 gl 4 Q Injection Mode Normal injection INJECT Cancel Sample Solution E Var by Cycle Volume jad Vary by Cycle Help Options Predip Needle Predip Solution IFredp Solution bw pole Estra Cleanup Injection W amp 2 vU 0 KEZET lt Normal injectian INJECT Normal injection IMJECT ptimized for speed minimum wash QUICKINJECT ptimized For kinetics including dissociation time KINJECT Dual injection COINJECT
75. Thermo A 2 MilliO R2F3 3ml BlAdesorb solution 1 R2F4 3 ml BIAdesorb solution 2 22 Desorb BIAdesorb solution 1 4C ur 1p yy J w ZcBupbcooUJblH unsge0c 0 d uU TU CAMAS ACC s 20 Sonitize MiliO9 Prime 1 2 D Sanitize 1 Sanitize BlAdisinfectant solution MilliQ 1 1 5ml BIAdisinfectant solution 20 ml 2 MilliQ 15 ml amp
76. 2 blm Program blr Result ble BlAevaluation software Ctrl Break 215 8161019 5 2000 Instrument Hondbook 30 3 3 2 5 5 Standby MAIN APPEND Standby C 3 Standby Running Standby Flows Remaining Time 4 00 i Stop stqndby Biacore 2000 Instrument Handbook 3 31 10 CD
77. Response Run Start New Cycle E Run Stop Sensorgram 14 10 306 5 10 30 Biacore 2000 Instrument Handbook 4 41 Binding of DSA 100ug ml to immobilized Asialofetuin 10000 4 Fc APRH G Time Window AbsRHesp 5D Slope Baseline REIResp 14 2851 2 0 27 0 05 Yeg Baseline 13243 015 0 07 Yes Baseline 13133 014 0 05 Yes Baseline 13424 3 0098 DD Yes Baseline 25755 4 69 27 36 959 bound 13255 85 043 0 21 Mo I bound 13422 5 0 0 DD bound Biacore 2000 Instrument Handbook
78. sNIA Toofastbinding of ligand ace in flowcells marked with has not been activated and may be used again 15 000 Biacore 2000 Instrument Handbook 9 Application Wizard 101 9 2 2 Specify Flow Rate and Injection Time wizard EX P Es NHS 5 pl min 7 5yl min 7 Ju immob Immobilization Frocedure Immobilization Procedure Aim for Immobilized Level Help lt Back gt
79. BIA programs Biacore 2000 Control Software MEIBIACORE 2000 Control Software Cw BIACORE 2000 Control Software on COMI Epitope Sensorgram zm Eile Edit View Command Run Tools Window Help Service4 la x lW E 2 Sensorgram Fc 4 Y RU Primary zMab 1 Second Mab_1_2 3 2 25000 Menu bar 24000 ool bar un A aa 7 22000 1 2 21000 d 20000 Y Sensorgram window 19000 18000 17000 16000 0 200 400 a00 1000 1200 1400 Time 5 Fc APROG Time AbsResp Slope Baseline RelResp Id Primary Secondary 1 epitop 47 5 18551 6 0 BASELINE _1 2 3 4 2 epitop 47 6 18401 0 0 05 Yes 0 BASELINE Mab Mab 1 _ 2 3 4 3 epitop 47 7 18782 1 0 02 Yes 0 BASELINE Mab 1 Mab 1 2 3 4 4 epitop 47 8 185143 0 03 Yes 0 BASELINE Mab 1 Mab 1 2 3 4 Reportpoint table 1 epitop 185 5 20741 5 0 00 Yes 2189 9 primary Mab 1 Mab 2 3 4 Event Log Sensorgram 4 Time Info Time 1377 8 s Response 18552 0 RU 0 0 Timestamp 29 May 95 13 35 25 Flow 0 mmin Flow Path e 0 0 SetTemperature 25 0 0 0 Temperature 25 06 0 0 Flow Cell 1 2 3 4 Temperature 25 00 C Detection Mode Single 0 0 Flow 5 p min Event log Table 0 0 Data Collection 1 Hz 56 2 Inject Start Fc 1 2 3 4
80. Sensor chip d Dock Dock Sensor chip Biacore 2000 Instrument Handbook q do qeu sega 1 Dock C dq sea 2 0 2 Undock Sensor chip Dock Jeden d ge 1 9 PEL DR cule scc Research Grade 15 Certified Grade
81. gt lose Mass Transfer Next gt Biacore 2000 Instrument Handbook 9 Applicotion Wizard 129 Kinetics Assay Principle x E ras irelreitsis Direct Binding Next gt Binding Using Capturing Molecule Kinetics Mass Transfer Control x Next gt i Biacore 2000 Instrument Handbook 130 9 Wizard Kinetics Regeneration Next gt a Kinetics Rack Positions Posion VoltD Conten R2 A1 320 50 Hm 1 Antigen m 195 1Hmll THERMO C REAG THERMO A a 8 e e e e e e e m 32 b 66 Next gt Biacore 2000 Instrument Handbook 9 Application Wizard 131 33i K
82. Stqndby Flow After Run Prime Before Run Start es Sawe Wizard Results As Save in E demo Es File Binding Analysis cave as type Results P hir gt Cancel Save Biacore 2000 Instrument Handbook 9 Application Wizard 121 KRIJ oc BA NO d 2 358 REGE D ne Tio bm File Edit View Command Tools Window Help x T1 pei ed y E Cycle 1 Curve Sensorgram Fc 3 RU 51 Repl 1 15200 15000 14800 14600 14400 14200 14000 Response 13800 13600 13400 al 100 150 200 250 200 250 400 450 Time Fc FPRD0 Time AbsResp Slope Baseline HelFesp Id Sample Analyse 51 5 149179 Baseline Analyse 111 5 1540153 0 24 dr 4 Sample Hespl 51 3 Analyse 262 5 149725 0 05 Mo 55 0 Sample Respz 51 Analyse 401 5 148898 0 12 28 1 Baseline Shift 51 Sensorgram Fc 3 Time 07 5 Hesponse 148894 RU Timestamp 99 06 04 19 22 46 Set Temperature 25 0 JIC Flow Flow Cell Temperature 25 00 JIC _ Rack 1 Thermo C Hack z Right Thermn Instrument Offline Flow Call 3
83. 135 NHS Empty vial tar EDC NHS Mix min capacity 200 B POEA Full ial Ligand Buffer 5 Ligand THERMO M Mane v THERMD_A M O OOO O OOO O OOO O OOO O OOO OOOO OOOOOOOOO OOOOOOO oOCOOOOOOO B ee eo 0000 O e sure that the correct rack s are installed In the autosampler when pou start the wizard run Help M lt Back Nest gt LIose Next gt R2A1 PDEA 4 5mg in 250ul 100mM Borate buffer pH 8 5 1 9 R2B1 UJ F RA 8 R2B2 R2C1 L cystein 1 5mg NaCl 14mg in 25001 0 1M Sodium formate Buffer pH 4 5 R2E1 EDC R2E2 NHS R2E3 NHS EDC Biacore 2000 Instrument Handbook 180 9 Application Wizard a Ligand Thiol Coupling blw Prepare Run THERMO 4A right hand rack position Position Volume pl Content 1 El PDEA B1 Full Ligand Buffer 2 T5 Ligand C Cysteine tatl 1 135 EDC E2 135 MHS Empty vial tor _ _ capacity 200 Lil Prime Before Run v Standby Flow After Run _ Hep
84. 200 ul 7mm 25 20C 1 4 200ul Biacore 2000 Instrument Handbook 3 19 5 200 ug ml 10mM pH 1 2 pH 21 86 pH 10 015 pH3 5 SA 0
85. s s s s s s s s s s st s s s s s s s tg s s tg t9 ng ng ng n 44 e 3 rYH F 0 e 1 s s s s s s s s s s s om om om om om on nm nm nm ng 52 ee 52 s s s s s s s s s s s s tttm ng 38 ESE s s s s s s s s s s s s s s A7 s s s s s s s s s s s s s e s s s s s s s s s s s s s tg tg s s tg ng ng ng 1 17 165 176 s s s s s s s s s s s s e s s s s s m m n m n n n 19 LI s s s 9 s 9 s s s s s s s s s s 62 OO 49 s s s e s s s s s 9 s s s 9 s m m om om tm m tm t t ng 45 s e s s e s s s s s 9 s s 9 s s 9 9 s 9 9 m 9 t n 1 75 J lt Dd Z gt s s s s s s s s s s s s s s s s s s s s s s s s o s s s s 10 EER E s s s s s s s s s s s s s s s s s st s m m s m s n n n n n ng 31 159 165 19
86. Injection Start window 5 s Cancel 1 baseline m Help Baseline Before Time 10 ld boseline 145 0 Baseline OK Biacore 2000 Instrument Handbook 146 9 Application Wizard INJECT END lgG i TEMP 25 DETECTION 2 1 e Ju Report Point Time 20 s s Injection End Window 5 s Cancel ld bound Hep Baseline After Time 20 Id bound OK INJECT END 10mM Gly HCIpH2 TEMP 25 DETECTION 2 1 F FLOW 20 10 BASELINE baseline 5 INJECT IgG 40 INJECT END 20 RESPONSE bound 5 4 INJECT 10mM Gly HCI pH2 20 gt INJECT END er 1201 Biacore 2000 Instrument
87. 75 77 Biacore 2000 Instrument Handbook 3 25 61 FLOW 10 10uMmin DILUTE R2B1 R2B2 R2B3 50 R2B1 R2B2 R2B3 EDC NHS 200ul 50 R2B1 50 INJECT R2B3 70 alga GL 72 2 7 7 CM 40 10 INJECT R2A1 70 R2A1 70ul 7 25 30ul 100u
88. Biacore 2000 Instrument Handbook 168 9 Application Wizard Next gt a Aldehvde Coupling bl Cycles Number of Cycle of Run 1 Next gt Dg za Aldehwde Coupling blw Detection Fc Next gt Biacore 2000 Instrument Handbook 9 Application Wizard 169 T Aldehyde Coupling blw Rack Positions Position vol 1 95 5 mi Hydrazine Ethanalamine Ligand Buffer Ligand Cvanoborahydride sure that the correct rack s are installed in the autosampler when pou start the wizard run Help THERMO M Mane v THERMD_A M O OOO O OOO O OOO O OOO O OOO OOOO 55 Haeneneratio OOOOOOO OOOOOOOOO OOOOOOOOODO 0 eii O e e Menu M lt Back Nest gt LIose 2 260 gt 27 70073585 2 1 R2A2 R2B1 2 1 R2E2 2 3 R2EA R2F1 R2F2 UJ UN PILT 8 C 50ug ml 0 1M Na cyanoborohydride 0 1M Na acetatebuffer EDC NHS
89. Ni 7 C18 monolayer 8 11 2 bilayer Biacore 2000 Instrument Hondbook 1 5 1 2 2 Prime Dock Working Tools Prime BA working Tools E 2 201 d EEA Start amp 2 U v 29d i Start Prime lt 19 Exit 2000 Instrument Handbook 6 1 2 Prime Tools Working Tools Prime
90. an injection of ESA Print Edit ma Chip Goupling blw Sequence Injection of liposome after surface wash Stabilization of the lipid layer with high flow rate and injection Check degree of coverage of the surface with an injection af BSA INJECT Detergent 25 INJECT END 5 EsxTRACLEAN F FLOW 2 BASELINE Baseline 5 INJECT Liposome Bl g amp l INJECT END F FLOW 100 El Help Menu gt Close Wizard Template Next gt Biacore 2000 Instrument Handbook 9 Application Wizard 173 Chip Coupling blu Cycles Number of Cycle to Run 1 Next gt ar HP A Chip Coupling blm Detection Made Fc1 Next gt Y Biacore 2000 Instrument Handbook 174 9 Application Wizard Ti Chip Coupling blw Rack Positions Position Vol il Content Rg A b5 Detergent Rg B1 120 Liposome 65 0 1 mg ml BSA 200 50 ml MaDH THERMO M Mane _ KK K
91. o o 6 3 3 E Extra Cleanup WW De SL We NULL NV US DL ND uo Ee ef ou TX CODO Dv X DS Se Ue 45 F We usu e M Sh apris rd vec NP Cm e uno te ts 53 e o 4 I If Then Wizard Wo Wo c ue DE WO IG S RC AMO VEU ORO oar NU UC OC veh uc cO Ql LAC CAD ES UNO UE 155 Immobilization Wizard QW 30131 701027201000 Ww 92 Immobilization pH scoutinglWizard 6 9 8 e e 86 INJECT 39 Biacore 2000 Instrument Handbook Kinetics onqlysis Wizord 122 KINJECT gt gt o e e e 39 L Linked Reaction Wizard W cd LEA Eo S CE QU cw der GE M CS EV oo 133 l5 e o 4 M Main SO 9 a QQ 61 Transfer Wizard De DE LES es 795 049 QUY WI CRT CQ aee UNS SO 16 o LE ec 97 9 c9 128 Normalize Mec rus
92. Acetate pH 4 50 Trypsin inhibitor ImmobPH 13047 5 8 Baselineb Trypsin inhibitor ImmobPH 18542 7 2 10 mh Acetate pH 4 00 Trypsin inhibitor ImmnhFH 13408 4 PreHegen Trypsin inhibitor ImmabPH 12835 8 6 Regeneration Trypsin inhibitor P m2 mam Poa mim nm PQ F Biacore 2000 Instrument Handbook 9 Application Wizard 91 ea ph4 Results Trypsin inhibitor 1 Acetate pH 6 0 nz SHE 10 Acetate pH 5 5 DE 10 Acetate pH 5 0 1000 10 mM Acetate pH 4 5 10 Acetate pH 4 0 15 4 32 4 48 56 Bi 72 Time s Ligand Butters verlay Plot 10 mM Table Immobilization pH pH RU pH pH RU Biacore 2000 Instrument Handbook 92 9 Application Wizard 9 2 Immobilization
93. R2B1 R2B2 R2B3 R2B4 R2B5 INJECT 40ul2 10mM Gly HCI pH2 20ul1 R2F3 mouse IgG 10 baseline mouse IgG 20 WIT bound baseline 60 regeneration baseline Biacore 2000 Instrument Handbook 160 9 Applicqtion Wizard 2 wizard 1 Ng ee Zea Keyword S OK Cancel E Help Keyword Analyte OK Conc Zea Keyword Kepword Canc Cancel 2 DETECTION 2 1 9 KETW RD E KEYWORD Conc F FLOW 10 KINJECT Sample ID 40 120 amp Biacore 2000 Instrument Handbook 9 Application Wizard 2139 L fc 57555 Marmal injection 2 El LI Fredip Solution Solution Keyword Analyte
94. Rack Base a evoca menvoa z Mone R MICRO THERMO_A du THERMO B CITHERMO C OK 8 2000 Instrument Handbook 9 JJ RS 3 Rack Base1 Rack Base2 e Thermo A 7mm 9 mm OS vd ph S 16mm Thermo_B 9 mm JE e Thermo C 2ml s lt rep A MICROS6 00D v4 IS V 7bi s BE TS Rack 1 Rack Base Id a 1 3 R1A1 R1A2 R1A3 2 R2F2 9670
95. Second Minute 3 C N Program Files BIACORE 2000 NGuide Methods Id Bia Users Biacore 2000 Instrument Handbook 3 27 3 2 3 Run Prerun Method OK gt gt Expected 1 2 3 4 1 2 5 4 2 1 4 3 2 1 3 1 4 1 2 1 1 2 DETECTION 5 5 Prerun Method HE 8 O
96. Stqrt Injection Biacore 2000 Instrument Handbook 22 3 T3 Injection Injection in Fc 1 Sample 2al v alume T Contact Time Consumption Mare gt gt Precnncentration of mouse IgG T pH m e Response mouse lgG Run Stop sensorgram 6 JUJSVbtV7bkv asvESRA TSEWEBDROpH pH NHS pH4 pH5 NH pH8 5 pH
97. 42 4 HE 11 pH 08 lt 1 10mM Gly HCI pH 1 5 HCI 100mM Phosphoric acid 100mM Formic acid 2096 10mM Gly NaOH pH12 NaOH 100mM Ethanolamine 100mM Ethanolamine HCI 1M EDTA lt 0 35M Surfactant P 20 Tween 20 lt 5 Triton X 100 596 SDS 0 590 Octylglucoside 40mM Acetonitrile lt 20 DMSO lt 8 Ethyleneglycol in HBS buffer 5096 Ethanol 2096 Formamide 4096 Guanidine HCI 5M Urea aM Biacore 2000 Instrument Handbook 4 43 12
98. Biacore 2000 Instrument Handbook 32 3 3 3 MANUAL INJECT NHS INJECT MANUAL INJECT Run Run Sensorgram MANUAL INJECT Command Queue 9 lt T Command Queue FIE Edt Hun Method File Close BIACORE x Closing the command queue will clear all commands in the queue Do want to close the command queue Ves Biacore 2000 Instrument Handbook 3
99. INJECT R2B4 70 R2B4 1M 70ul NHS amp EDC NHS 30ul 1000 Biacore 2000 Instrument Handbook 26 3 3 2 2 MM Edit Adjust Method Second Minute define qprog immob caption immobilization flow 5 dilute r2b1 r2b2 r2b3 inject r2b3 35 0 10 rpoint baseline b end Edit gt Adjust Method Minute DEFINE APROG immob CAPTION immobiilization FLOW 9 DILUTE r2b1 r2b2 r2b3 INJECT r2b3 35 0 10 RPOINT baseline b END 7 D 1 D
100. MAIN MAIN Run MAIN DEFINE APROG Analysis Program 088 o ccc 9 e 29 2 09 1 DEFINE APROG injection FLOW 5 INJECT R2A1 5 END FLOW 5 l min DEFINE APROG injection injection DEFINE APROG Run Prerun Method error Prerun Meth d Ix 27 Biacore 2000 Instrument Handbook 8 63 gt gt Expecting keyword MAIN MAIN DEFINE APROG
101. MAIN FLOWPATH DEFINE APROG FLOWPATH DEFINE APROG DETECTION MAIN 1 2 ele d 1 2 3 1 2 3 4 1 2 3 2 1 3 1 4 1 1 2 3 4 1 2 3 4 1 2 3 4 1 2 1 3 1 4 1 1254 7L UJb 1309 7 w b 7415 955 2 1 4 3 MABE 123 1234 DETECTION 123 9 1 4 1 UN AU Biacore 2000 Instrument Handbook 70 8 KDRP CX Cc 589 SC CD FLOWPATH FLOWPATH FLOWPATH FLOWPATH FLOWPATH FLOWPATH FLOWPATH DEFINE APROG path FLOW 10 FLOWPATH 1 FLOWPATH 2 FLOWPATH 3 FLOWPATH 4 END MAIN DETECTION APROG END
102. OK FLOW 20 FLOW 20 ise elg emma t 3 a TEMP 25 DETECTION 2 1 FLOW 20 Biacore 2000 Instrument Handbook 144 9 Wizard DETECTION 2 1 F FLnw 20 INJECT IgG 40 amp INJECT END DETECTION 2 1 T Inject Fredip Solution En i 2 OK i Biacore 2000 Instrument Handbook 9 Application Wizard amp fc FB L c 32825 gt TEMP 25 DETECTION 2 1 F FLOW 20 INJECT 40 INJECT END INJECT TUmM lp HLLnHg 20 A g amp l INJECT END INJECT IgG 40 ac TEMP 25 DETECTION 2 1 F FLOW 20 g INJECT END Ee 7 es mm Report Point Time 10 s C
103. Save As Save Results As Save n Eg Data File name precancentratiorl Save as Results bir Cancel Save in C C NBia UsersN AJU Save Biacore 2000 Instrument Handbook 3 1027 17 3 90 1 CM5 CM5 Se N e E CM NHS N
104. b RPOINT bound INJECT R2F3 20 RPOINT regeneration RACK 1 thermo b RACK 2 thermo a DETECTION 2 1 APROG assay R2A1 Antigen 25ug ml APROG assay R2A2 Antigen 12 5ug ml APROG assay R2A3 Antigen 6 25ug ml APROG assay R2A4 Antigen 3 125ug ml APPEND standby c0 7D 7 2140 20 021 8120 bei 1 NS DETECTION DETECTION DETECTION DETECTION 12 DETECTION 2 1 DETECTION DETECTION 1 2 5 DETECTION 1 4 3 DETECTION DETECTION 2 1 3 1 4 1 DETECTION Biacore 2000 Instrument Hondbook 4 49 4 2 2 26 4 2 3 27 4 2 4 28 Binding of DSA 100ug ml to immobilized Asialofetuin 4 2 5 5 30 28 Ctrl Break
105. bound 50RU AA KS C FARDD Eg es pe IF FielFiesp For Report Paint bound i Flow Cell 2 pu A3 B Comparison ls Less Than Cancel Help Constant walue 5n Hep Use Additional Condition THEN Exi Cycle Biacore 2000 Instrument Handbook 156 9 Applicqtion Wizard Report Point 00017 5 XR RelResD AbsResD Slope RU s In Flow cell 9 Z T U Comparison EBC NS 5107260008 Is Greater Thon Is Greater Than or Equals Is Less Than Is Less Than or Equals Equals THEN Exsit Cycle Exsit Method Command Block If Then
106. 17722 1 0 01 Fc Ligand 4 71 5 16878 72 85 14 3 antialb 1Dug ml 911 0 1030 1 Aimed for 1000 RU 3 Aim4 775 174274 96 14 3 Aim4 91 5 188049 100 47 3 Aim4 121 5 21993 7 113 46 3 Aim4 166 5 26513 3 73 43 i 17706 2 0 01 El s Response 18737 6 RU Flow Path 00 C Detection Mode Single 0 Timestamp 16 Oct 38 16 39 10 0 0 Set Temperature 25 0 C 0 0 Temperature 25 00 0 0 Rack 1 Left Thermo C 0 0 Rack 2 Right Thermo 0 0 Rack Reagent REAG Immobilization Surface Performance Test 40000 37000 34000 31000 28000 25000 Ethanolamine Ligand 22000 NHS EDC 19000 T 16000 i En Response 2 13000 Ea der 10000 f i 200 400 600 aD 1000 1200 1400 1600 1800 Time 5 Response 1 RU Response 2 NHS RU 100080 Response 1 Biacore 2000 Instrument Handbook 100 9 Application Wizard 22 wizard DPA Wizard NHS
107. Biacore 2000 Instrument Handbook 24 3 pO DP ban E A DEFINE APROG immob CAPTION Amine coupling FLOW DILUTE INJECT RPOINT INJECT INJECT RPOINT RACK 1 RACK 2 10 R2B1 2 2 R2B5 50 R2B5 70 Baseline b R2A1 70 R2BA 70 immob thermo b thermo a DETECTION 2 APPEND immob standby 10ul min NHS EDC NHS EDC E immob Stqndby R2A1 EEA pH R2B1 NHS SL ld 7S48I87J RV U BB US REB T 0D 6 02 R2B2 EDC GL X I amp NHS 7 O ELU p L8 REIB S 00 6 0 R2Bs 3 7 mm R2B4 UE N 30 1 4
108. Id mh E 1 kimelic 595 173341 2 D 258 res 0D HASELINE 285 Pa to p d AI nag v o 16305 asd 77 aa 1 kinetic 646 5 192212 0 39 es 149 6 analyte 286 1 kinetic 376 5 175954 0 16 Mo 1625 9 BASELIHE2 286 Response Event Log ime Info 00 Timestamp 95 05 17 08 59 44 00 Set Temperature 25 0 Flow Flow Cell Temperature 25 05 z 0 0 Flow Cell 1 2 3 4 Taanit L HH Flow 20 I l min Instrument Offline 0 0 Data Collection 1 Hz Cycle 1 Eom 1 eccle Biacore 2000 Instrument Hondbook 5 51 5 5 1 2 1 Standby 3 Standby 3 SS 21795 Standby Tools gt Working Tools Standby 5 ul min 96 02 7
109. R1B1 Menu bor Biacore Toolbar Sensorgram window pou xe Js Zl seme Report point table Eventlog window X S EO Status window RU Run Biacore 2000 Instrument Handbook 1 3 1 2 1 2 1 Dock Dock Da Dock EJ Insert a new sensor chip Click x Dock when ready After docking should run Prime from Working Tools FONA 27 A7 lllcS X Cl C C5 Ml Ce 2
110. Run v Standby Flow After Run Menu lt Back Start Llose Standby Stqndby Flow After Prime Prime Before Run Start Biacore 2000 Instrument Handbook 90 9 Application Wizard Save Wizard Results As Save in 3 Demo T File name pH Scouting Save as Results blr Cancel Sqave SEBRIJA 4 SERIE S 9c RU Immobilization pH Scouting 20000 19000 12000 17000 16000 15000 14000 13000 12000 11000 10000 Time lt Slope Baselne ReIREsp ImmobPH 12874 8 Baseline Trypsin inhibitor ImmnhFH 11153 6 2 10mh Acetate pH 6 00 Trypsin inhibitor ImmnhFH 12872_ 5 7 Baseline Trypsin inhibitor ImmobPH 11150 2 10 mh Acetate pH 5 50 Trypsin inhibitor ImmobPH 12874 1 5 Baseline3 Trypzin inhibitor ImmnhFH 11153 8 3 10 mH Acetate pH 5 00 Trypsin inhibitor ImmnhFH 12873 7 4 Baseline4 Trypsin inhibitor ImmnhFH 16126 0 3 10
111. UK K GG K e 9o 99 006 sure that the correct rack s are installed In the autosampler when vou start the wizard run Help Menu M lt Back Nert gt Llnse Next gt B eS 1 og 0000 O O O O LI O K K K K u K N EE EE O e e R2A1 R2B1 R2C1 0 1mg ml BSA R2F3 50mM NaOH Biacore 2000 Instrument Handbook 9 Application Wizard 175 2 Chip Coupling blw Prepare Run kl THERMO A right hand rack position Position Volume pl Content A1 B5 Detergent H1 120 Liposome MN B5 0 1 mami B F3 200 5 Prime Before Fun ly Standby Flow After Run _ Heb Menu lt Back Start Close Start Ssqve Response Time AbsHesp Slope Baselime RelIResp 3296 5 112130 001 Yes baseline 3868 5 114904 0 18 277 5 20mM Chap 5825 5 124737 0 12 Mo 1250 8 Hm liposome 6157 5 122533 0 08 1040 3 1 mM NaOH Biacore 2000 Instrument Handbook 176 9 Application Wiz
112. minimum capacity 200 HI 4 a5 Fetuin CS 75 Ethanalamine Prime Before Run W Standby Flow After Run _ Heb Menu lt Back Start Close Stqndby Flow After Prime Before Start Save In J Demo File name immobilizatior Bave Save Lupe Fiesults bir bd Cancel Save Biacore 2000 Instrument Handbook 104 9 Application Wizard Amine Coupling Fetuin 3 FlowAndTime 286 5 0 122008 011 0 0 Baseline No 2 Fetuin 3 FlowAndTime 839 5 0 123129 0 16 0 112 1 EDC NHS No 2 Fetuin 3 FlowAndTime 1088 5 0 15533 7 1 17 0 3333 0 Ligand No 2 Fetuin 3 Flow ndTime 1638 5 0 15417 6 0 18 0 3216 8 Ethanolamine No 2 Fetuin 3220 8 3218 8 Injection time 2 0 min flow 10 min Response 1 2 99 Biacore 2000 Instrument Handbook 9 Applicotion Wizard G f FBU 105 9 3 Reqeneration Scouting
113. position Position Yolume pl Content c 115 Coupling solution C2 115 MHS Coupling Solution Empty vial for mixing minimum capacity 200 pil 4 210 Antibody 75 Ethannlaimirie c10 s 5 mM NaOH Prime Before Run e Standby Flow After Run Heb Menu lt Back Start Close Standby Flow After Run Prime Before Run Start Save In J Demo File name immobilizatior Save Save as Hype Fiesults bir m Cancel Sqve i Biacore 2000 Instrument Handbook 9 Application Wizard 99 73 BIACORE 3000 Control Software on COM immob_anti alb Sensorgram hm File Edit view Command Run Tools Window Help 18 x bri al Cycle 1 v Curve Sensorgram Fc 3 RU anti alb f ug ml target 1000 RU 34000 32000 30000 28000 26000 24000 Response 22000 20000 18000 16000 34 immob anti alb Results 2500 Fc APROG Time 3 Aim4 Response 1 Hesponse 2 Procedure bound final AbsResp Slope
114. s s s s s o s s s s s o s s s s tg n 9 9 9 t t n 1 53 s s s s s s s s s s s s c 9 s 9 m m nm 14 n ng ng n n 43 e s s s 55 Biacore 2000 Instrument Handbook INDEX A Aim for immobilization levellWizord s ce ce 95 Application Wizard e e 84 B C gt gt s e e e e e o o e o 39 Binding Analysis 0 116 Close s s s s e e o e e o o so o o s o o o s o 51 CM5 RU DG ILS WU WO QS UN CAD e 8 RE Tr eu GS US le le 09 Ue Uaec DEL RIS Ee SU 19 4 COINJECT S gt gt gt e e e e o 59 Command Queue LN ELE E 46 Control Experiments Wizard 128 Customized Application Wizard e 9 9 9 9 138 D Desorb s We n4 St Q Q ie Wo EC r Io WI CUM Cd 9 re iW CISCO OQA SS U o L o DLL NI I D xe 59 5 S CN 54 Dock e wc w o
115. 22 0 12 117 2 EDC NHS anti alb 1 ug ml 1000 3 Am4 1611 5 18734 2 0 02 No 1028 3 Ligand 10 1000 3 Am4 2161 5 187353 0 19 No 1030 3 EIhannlamine anti alb 1 0ug ml 1000 Application Wizard 95 A Run Stop Sensorgram 92 Biacore 2000 Instrument Handbook 4 3 7 4 crude
116. 4 Command gt Inject Biacore 2000 Instrument Handbook 4 39 T3 Injection Injection in Fc 1 l mauwa INJECT Cancel FKIMJELT E OUICKINJECT COINJECT BIGINKJECT MANUAL INJECT Contact Time Consumption gt gt INJECT INJECT 5 325Ll 30Ll ss COINJECT 2 Sample 1 0 100yl 40yl Sample 2 0 100Ul 40yl Zea Injection Injection in 1 KIMJECT Sample Cancel za V alume Contact Time Consumption Dizzociation Time gt gt Start Injection eU weg Biacore 2000 Instrument Handbook 40 4 42 L INJECT
117. Close Specify Flow Rate and Injection Time Next gt sm immob Flow Rate and Injection Time Flow Cell Ligand Hame Time Flow lt Back Hent gt Close Next gt Biacore 2000 Instrument Handbook 102 9 Application Wizard a Surface Preparation Rack Positions Position Vol f 115 EDE Coupling Solution 115 NHS Coupling solution Empty vial far mixing min capacity 200 Fetuin 5 Ethanalamine THERM 9 9 555900 Jooeoo OO To Any changes vou make to the rack settings here will not take effect until vau run the wizard Hep M lt Back Save As Close Next gt Es Biacore 2000 Instrument Handbook 9 Application Wizard 103 Ta Surface Preparation Prepare Run THERMG A right hand rack position Position Volume pl Content 1 115 EDC Coupling solution 52 115 MHS Coupling Solution Ca Empty vial for mixing
118. GE Healthcare 3 Biacore Biacore 2000 Ver 5 Instrument Handbook GE imagination at work 71 3201 31 1 1 1 2 qaqa qaa qu a ua ut eet ttn 1 1 1 1 80175 FIJA 7 1 ume JJ p 2 FR UN nerunt x MM uyanman naman 3 1 2 1 Dock 2 46 3 1 2 2 Prime 5 i Me Lib URN li pong n c D DUI MUN E NM URS 9 1 2 5 SPR Normalize 10 2 11 ra ONAN od Gi 11 2 2 14 D MEAM c Em 16 23 SOS ud hu 17 x1 S 72 22 20 3 2 JOZ JN TTE IC U JJ orci ertet tct 23 a a ETETE EAEE E N A E 23 2 26 LG ON A 27 CR NEP RU PR UD 28 ueri DIAS a EE 30 3 3 ela L 73 73 5 08 32 4 37 4 1 4 38 4 2 44 47 ace sed 47 49 Moe ccm 49 49 5 m Ae D M RR 49 CES LADEN CLIP e E E A SDpDpDRARcRR R cRCCRRRRAARRAAAAAj jj 51 A a
119. H 4 00 140 50 mM NaOH THERMO C REAG THERMO t ooo eeeele se o 555 eoooOO Make sure that the correct rack s are installed in the autosampler when you start the izard run Help Menu M lt Back Nest gt Close wizard Menu gt Template Save As 93 Nest gt 89 ph4 Prepare Run x THERMO right hand rack position Position Volume pl Content A1 50 Ligand in 10mM Acetate pH 6 00 A2 50 Ligand in 10 Acetate pH 5 50 50 Ligand in 10 mM Acetate pH 5 00 4 50 Ligand in 10 mM Acetate pH 4 50 5 50 Ligand in 10 mM Acetate pH 4 00 F2 50 50 mM NaOH Prime Before
120. Handbook 9 Application Wizard 147 Zea Report Point x EA Time 0 s a Fd Injection End window 5 s Cancel fregeneratio 0 Help Baseline OK Ces HRD a TEMP 25 2 DETECTION 2 1 F FLOW 20 jpg k BASELINE baseline 5 INJECT IgG 40 INJECT END 20 RESPONSE bound 5 F2 INJECT 1Umk HL pHZ 20 INJECT END 2 RESPONSE regeneration 2 Next gt Ded Customized Cyclez Humber of Cycles to Bun Help Menu hu lt Back gt Close Biocore 2000 Instrument Hondbook 148 9 Application Wizard 1 Next gt 1 Rack Positions Hi Di 95 1005 Ri D2 75 1 mM Gly HCI pH V IgG 10mM Gly HCl pH2 00 VA 7 2058 9 227589 esl as Biacore 2000 Instrument Handbook 9 Application Wizard 149 aa 1 Prepare Run E THERMO A right hand rack position Position Volume pl Content 50 IS F3 170 1 1 Gly HCI pH2 Prime Before Run Iw Standby Flow After Run Hep M
121. INE APROG immob CAPTION Amine coupling FLOW 20 DILUTE R2B1 R2B2 R2B3 50 INJECT R2B3 70 RPOINT baseline b INJECT R2A1 70 35 20 10 INJECT R2B4 70 RPOINT immob DETECTION 1 2 3 4 APROG immob APPEND standby Biacore 2000 Instrument Handbook 8 79 6e 2 R2A1 DEFINE APROG assay FLOW 20 INJECT R2A1 40 END MAIN DETECTION 1 APROG assay APPEND standby END 3 R2A1 R2A2 R2A3 DEFINE APROG assay PARAM pos FLOW 20 INJECT 40 DETECTION 1 APROG assay R2A1 APROG assay R2A2 APROG assay R2A3 APPEND standby Biacore9 2000 Instrument Handbook 80 8 Bldcore 0D 2L 24 0
122. Instrument Handbook 4 47 4 2 Assoy bim 61 Application Wizard L 116 2 122 4 2 1 2 File gt 2 72 795 BIACORE File Look in E Methods Gradient Kinetic a L thiol E Recover 2 Epi S thiol Fraction File name Files af type al BIA Files bl Cancel C NProgram FilesNBIACORE 2000NguideNMethods Open ge Biacore 2000 Instrument Handbook 48 4 DEFINE APROG assay PARAM 96pos ID 96conc CAPTION Binding of ID conc to immobilized antibody FLOW 20 FLOWPATH 12 KINJECT 40 120 RPOINT baseline
123. L 03 5331 9336 FAX 03 5331 9370 e mail Tech JP ge com
124. M Glycine pH 15 1 00 Regeneration 209 Analyte 80 2 801 lt 9 5 pH 20 80 Analyte 80 20 2 90 Biacore 2000 Instrument Handbook 9 Application Wizard 111 9 4 Surface Performance Test wizord 1 2 Run Run Application Wizard Surface Preparation Start Surface Performance Test Next gt l i Surface Preparation Analyte Settings E gt Analy
125. Menu lt Back Start Close Standby Flow After Run Prime Before Start Save Bs 1200 1400 1600 1800 Fc APRDBR Time AbsHesp Slope Baseline HalHgsn Id 2 thiol 2835 103207 0 08 Yes Baseline 2 hml 17785 176168 0 0 99 Mo 7 2952 Immnb Biacore 2000 Instrument Handbook RI 7 291 77 00 10 om s o e e o s s o 67 0 90 jy e s e s s s s s s s s s s s s so s s o 0 0 s s c 9 9 9 37 s s s s e s s s s s t s s s s s tm t n s 9 n9 n 84 76 J 71 106 s s s s s s e s s s s s s s s t st m t m n t n n tg ng n 9 4 17 18 25 60 92 s s s s s s s s s 4494944 4 18 FJS e s s s s s s s s s s s s s s s s s s s s s s s s s s 9 9 9 9 4 17 92 165 166 DI H NR FY JL R s s s s s s s s s s s s s s s s 0 s s s s s s 9 8 165 55 T222ZFE22DIJ U Jo s s s s s s s s s s s s s s s s s o s 0 s 0 s 9 9 o 9 45 ID DAR s s s s s s s
126. OO OOOOOOOOOO OOO0O0O0O00000 CICOCOCOCOCOCOCOCUL Help Menu Next gt lt Back gt Close Sx Surface Preparation Prepare Run REAG reagent rack Position Volume pl Content 2 220 THERMO 4A richt hand rack position Position Volume pul Content 1 360 Antigen Prime Before Run 2 Standby Flow After Help Menu lt Back Start Llansg Biacore 2000 Instrument Handbook 114 O Application Wizard Standby Flow After Prime Before Run Start Save Results As 3000trainino Lit 1 1 we Pesen Sqve lolx 181 3 eu l RU Surface Performance Test ai regeneration test Results 40 3 Performance 64 5 18706 1 0 3 Performance 144 5 18869 2 0 Biacore 2000 Instrument Handb
127. P gt lt e5X33J2 lE Ty OFF TEL 03 5331 9336
128. Sl t gt ak Nes Cose 18071 OLeRSRHU SRMRERROXCCUX Biacore 2000 Instrument Handbook 9 Application Wizard 139 9 7 1 E jm om s a J 17 i 2 x F JEF POPTY K m ct a CUBOISTRIL FUN ee IFC 52 x AUS CD aas JSIR Then
129. Y n 150 110 70 30 1D I 50 Response 100 150 200 250 200 250 400 450 Time Apparent constants according to Fitting model 1 1 Langmuir binding 1 2e5 17M kd 4 4 1 HMax 338 AU 2 8688 17M KD 3 4 9 M Concentration Series Baseline Level 1 5 ka 1 5 1 M Ko M Rmax 1 1 binding model BIAevaluatlon Baseline Level cha rabbit IgG Results Flow Cell 1 Baseline Stability 14000 14074 14069 14062 14056 14050 14044 14038 14032 14026 14020 Response Concentration Series Biacore 2000 Instrument Hondbook 128 9 Application Wizard 9 6 2 Control experiments 9 6 2 1 Mass transfer wizard
130. a a e A A mus a a AAA 51 5 2 Undock ee 52 5 3 rA na EI c H 52 Biacore 2000 Instrument Handbook 6 53 ME ITT 7 55 qaqaqa qa aaa e 55 6 5 BRI sS rt doc c ex UD MI TS aissos 55 Bs CC TU Y uuu su aM EIUS 56 zm 9 9 9 S 59 BB UH ecce 61 9 Application Wizard amp FH UL Jc SESS JJ 72s enne 84 9 1 Immobilization pH Scouting 86 9 2 Immobilization gt 92 9 2 1 Aim for Immobilized Level 95 9 2 2 Specify Flow Rate nn ec IM 101 9 3 B RE Regeneration Scouting 105 9 4 Surface Performance Testj 111 9 5 Binding analysis 116 9 6 Kinetic analysis 122 9 6 1 Concentration 122 9 6 2 Control experiments 128 9 7
131. ard 9 8 3 NHS EDC 10mM 80mM PDEA in 0 1M Borate bufferlpH8 5 50mM l cysteine 1M NaCl in 0 1M Formate buffer pH4 3 Zea Mew Application Wizard OUrface Preparation 555 Binding Analysis Kinetic Analysis Customized Application Create Mew Template Cancel qi The Surface preparation application wizard guides wou through the essential steps in preparing the sensor surface for analysis including immobilization pH scouting ligand immobilization regeneration scouting and surface performance test Open Template C N Program Files BIACORE 2000 Guide Method Wizard Template Ligand Thiol Coupling blw Biacore 2000 Instrument Handbook 9 Application Wizard 177 52 Ligand Thiol Coupling blw Wizard Template Wizard Type Customized Application Notebook Ligand Thiol Coupling method For immobilization of ligands bu intrinsic thiol groups referred to as the
132. au should run replicates af at least one sample per analyte 1 cda 227224 hkoOls EOUTUZ Vetdsiae U Ed cce Ignore Kinetics Regeneration Regeneration Flow Rate Flow Rate 30 min Regeneration Method f Dissociation in Buffer Single Injection C Two Injections Solution Injection S olution 10 mM Glycine pH 1 9 Injection Time 30 s Predip Needle Stabilization Time After Regeneration 2 min Help lt Back gt Close Nexty i Biacore 2000 Instrument Handbook 9 Application Wizard 125 Tu Kinetics Rack Positions an 10 pg ml 66 7 Rabbit IgG 90 5 33 3 nM Rabbit Igi Jn 2 5 ug ml 15 7 nM Rabbit lg 90 1 25 pg ml 8 33 Rabbit IgG 325 10 mh Glycine pH 1 5 see 2000090 dique eocooo e0000 ee eoooo0n0 Imooooo sure that the correct rack s are installed in the autosampler when vou start the wizard run Heb Menu M lt Back gt Close Next gt i 53 Kinetics Prepare Run THERMO A right hand rack position Position Vo
133. e Preparation Start Binding Analysis Kinetic Analysis i T Use T late Customized Application Use remplace Cancel qii The Surface preparation application wizard guides you through the essential steps in preparing the sengor surface For analysis including immobilization pH scouting ligand immobilization regeneration scouting and surface performance test D Surface preparation 5 1 Immobilization pH scouting U DY PEOR e ERI DEAD wizard 2 Immobilization wizard 3 Regeneration scouting wizard 4 Surface performance test wizard Binding analysis wizard PL x Mq Biacore 2000 Instrument Hondbook 9 Application Wizard 85 Kinetic analysis ko M kg MIs1 kg s1
134. ent Hondbook 136 9 Application Wizard T Kinetics Prepare Run x REAG A reagent rack position Position Volume pl Content 1 100 10 THERMO 4A right hand rack position Position Volume pl Content 1 3ru 50 1 pii Antigen Prime Before Run Z Standby Flow After Run Hep muera lt Back Start Close Standby Flow After Run Prime Before Run Start aM Save Results 5 Save n c 3 ntraining regeneration test ixi Fatuin zi immob anti alb ixi immob RAMRC mass tamsfer test mass tarnsfer test File name ink gave Save as pe Results Cancel Biacore 2000 Instrument Handbook 9 Application Wizard 137 Save 52 mass tarnzfer test Results EBSA BSA 10 160 140 120 100 Response 40 20 20 0 TRIN 1000 1500 2000 2500 Time Baseline Level Mass Transfer Linked Reactions SE to SR 1 1
135. enu lt Back Start Close Standby Flow After Run Prime Before Start Wizard Results Save n J Demo File name lg Save as Fiesults bir Cancel Sqve i Biacore 2000 Instrument Handbook 150 9 Applicqtion Wizard DETECTION 2 1 F FLOW 20 E A INJECT IgG 40 gi INJECT END A INJECT 10mM Gly HCI pH2 20 g INJECT END XIADA 071 9 7 des A DETECTION 2 1 F FLOW 20 37 INJECT laa Edit Command Ctri E g INJECT END Eeport Point Gtr HH F INJECT 10 Inject FH Comment tr liri Wait GT WW Transfer Gtr M Flow Path Flow Ctrl F Wazh If Then Settings treer Keyword cave Wizard Template Gtr S Save Wizard Template Az Print Sequence Print Wizard Template Delete Command Delete Comnmand
136. g ml to immobilized antibody AO 0 DEFINE APROG assay PARAM pos FLOW FLOWPATH INJECT RPOINT baseline b RPOINT bound INJECT R2F3 20 RPOINT regeneration DETECTION 2 1 APROG assay R2A1 APROG assay R2A2 APROG assay R2A3 APPEND standby Biacore9 2000 Instrument Handbook 84 9 Application Wizard 9 Application Wizard Wizard Wizard Wizard start Wizqrd Run Application Wizard surfac
137. inetics Prepare Run lx REAG reagent rack position Position Volume pl Content 2 135 1Hml THERMO A right hand rack position Position Volume pul Content A1 320 50 1 Antigen Prime Before Run Standby Flow After Run _ Heb Menu lt Back Start Close Standby Flow After Run Prime Before Start Save Results As in e ut assay immobilization precon File name masa gave Save as Lupe Results m Cancel Sqave Biacore 2000 Instrument Handbook 132 9 Application Wizard Ti mass tarnsfer test2 Results Analyte JESA BSA 100 140 120 100 Bn 5 ul min 50 45 40 Response T5 Himin 20 20 0 30 90 120 3150 190 210 240 270 300 Time s Baselne Level Mass Transfer Linked Heactions EOR 213 SERRTGSR TD ING TVs 5 JBAR8J SEAS BELEU HY E C mJUU 77772 EER
138. ise Short term Longterm Slope Fc 1 0 3 RU Fc 1 0 01 lt 0 05 RU s Fc 2 0 3 RU Fc 2 0 01 lt 0 05 RU s Fc 3 lt 0 3 RU Fc 3 0 00 lt 0 05 RU S error Biacore 2000 Instrument Handbook 7 7 S JE 7 59 Users My Computer Windows My computer Bia C Bia Users x File Edit ew Go Fawortes Hal Bark Hp Cut Bia lib Biaewal Check Compac Dema Dimid gt abject s selected My Computer Users MEEI Eile Edit View Go Favorites Help a Shnrtcut Delete 4 Wave Sound Bename Tex Document Properties wordPad Document
139. l run time is about 40 minutes NOTE This procedure should be run at 25 C with a new Sensor Chip CH5 Last Run Time Start amp 27 v 22 3 5 Biacore 2000 Instrument Hondbook GS TU UA 57 i 9 A A 500 1000 System Check To evaluate the System check click the Evaluate button 40 Evaluate Biacore 2000 Instrument Handbook BIACORE 2000 System Check Name Created By BIACORE 2000 Control Software Version 3 0 7 Beta Instrument id 2076 Configuration IFC3 with Recovery Run Date 22 Mar 1999 Evaluation Date 02 Sep 1999 Analysis temp 25 0 RESULTS A IFC Serial Sequential Fc 1 22165 19141 Fc 2 22469 21141 Fc 3 22501 21193 Fc 4 22461 21159 Fc 1 2 3 4 22439 Leaks 34 B Refractometer test 15 Sucrose Baseline level Fc 1 22209 Fc 1 15246 Fc 2 22510 Fc 2 15334 Fc 3 22538 Fc 3 15435 Fc 4 22499 Fc 4 15390 Average 22439 22000 23000 Average 15351 Stdev 154 lt 200 Stdew 82 300 C Mixing Mix 1 Mix 2 Average Difference Dilution factor D No
140. ligand thiol Save See Bl amp applicatiorris Handbook Far more infarmatian Print Edite TET 52 Ligand Thiol Coupling blw Sequence E Ligand Thiol Coupling method tor immobilization af ligands bu intrinsic thiol groups refered ta as the ligand thiol procedure See BlAapplications Handbook For mare Information TRANSFER EDL_ MHS Mix 100 TRANSFER NHS EhL NHS him 100 c MI EDL MHS amp utoMix p BASELINE Baseline 5 INJECT EDE NHS Mix 10 e e INJECT END NOTE Extra washing i recommended after LM pulses PDEA 20 A INJECT END Heb Menu lt Back New gt Wizard Template Next gt Biacore 2000 Instrument Handbook 178 9 Application Wizard Chip Coupling blu Cycles Number of Cycle to Run 1 Next gt 5 Chip CGoupling blu Detection Fc Next gt Y Biacore 2000 Instrument Handbook 9 Application Wizard amp fc FB L 52825 sa Ligand Thiol Coupling blw Rack Positions Position Vol i contenr
141. ls ZAN FIaw Hate 20 Humber Sample Injections 1 E Sample Injection Time 2 min Walt After Injection 2 2 min Help lt Back Hest gt Close 10 20ul min EE FJ ZA 0 573 Next Wait After Injection 2 5 Number of Sample Injections 4 2 3 1 Biacore 2000 Instrument Hondbook 118 9 Wizard Zea Binding Analysis Cycles Bun Order n Entered TO 9 151 1152 154 155 156 1158 1159 lt Back gt Clase Run Order Entered Random Ste Random As Entered
142. lume pl Content 1 an 20 133 nM Rabbit Ig A2 an 10 66 7 nM Rabbit IgG A3 g0 5 uqi 33 3 nM Rabbit Ic A a 2 5 pg nl 15 7 nM Rabbit Ig AS a 1 25 8 33 nM Rabbit Iac F3 325 10 mbl Glycine pH 1 5 Prime Before Run Standby Flow After Run _ Hep Menu lt Back Start Close Stqndby Flow After Run Prime Before Start Biacore 2000 Instrument Handbook 126 9 Application Wizard Save wizard Results As Cave in E dema E save as type Results gt Cancel Save KRIJ c BA NO d ne Tio Rabbit InG 2 0pg ml 1 1 binding model Biacore 2000 Instrument Handbook 9 Application Wizard 127 rabbit IeG Results Analyte F abbit laa Rabbit 196 310 270 230 190
143. n preparing the sengor surface for analysis including immobilization pH scouting ligand immobilization regeneration scouting and surface performance test Open Template C N Program Files BIACORE 2000 Guide Methods N Wizard Template Aldehyde Coupling blw Biacore 2000 Instrument Handbook 9 Application Wizard 167 esi Aldehwde Coupling blw Wizard Template Wizard Customized Application Notebook Aldehyde Coupling method Far immobilization af ligands containing aldehyde groups to hydrazide modified supports The method can he used e g proteins with carbohydrates where aldehydes are formed bu osidation before coupling See Bl applcations Handbook For more Information TRANSFER HHS 100 TRANSFER NHS NHS His 100 Mis MHS Mix AutoMix k BASELINE Baseline 5 INJECT EDC NHS Mix 15 INJECT END DIPHEEDLE 5 mM Hydrazine INJECT 5 Hydrazine 35 g amp l INJECT END MOTE Estra washing is recommended after LH pulses INJECT END INJECT Ethanalamine 35 E EXTRACLEAM MOTE Predip needle used For sensitive ligands X DIPMEEDLE Linand Buffer Bee gt lt Back Neb Wizard Template
144. o 11514 1 8 sucrose 1 648 5 35304 9 0 92 No 22808 9 16 sucrose Stop Sensorgram 312259 5 Biacore 2000 Instrument Handbook 14 2 2 2 RU 2 28 View Reference Line Eo ege es i DUET i 8 Edit gt Add Report Point 5 5 Add Report Point Sensorgram F Time as 5 z Cancel window 5 Id baseline v Baseline E Apply to all curves ld
145. ol the interaction after varing lengths of time at steady state and presents the results as an overlay plot aligned ta the start af dissociation Help gt L lose Linked Reaction 23 L Next gt 7 UJ 795 i Biacore 2000 Instrument Handbook 134 9 Application Wizard i Kinetics Assay Principle Direct Binding Next gt Binding Using Capturing Molecule Kinetics Linked Reactions Control zix Next gt i Biacore 2000 Instrument Handbook 9 Applicotion Wizard 135 Kinetics Regeneration Next gt Kinetics Rack Positions Position Vol g R2 1 3 0 50 ug ml 1 uM Antigen m _ 1001bmhv THERMO A e e e e e FYTI t u FU Next gt amp 2 0702072952 Biacore 2000 Instrum
146. ook 9 Application Wizard 115 Baseline Level Response Level Baseline Level Response Level LOL 17 lt 157002118 sa 822103627 e o YS lt 21 LLA xD 709 Baseline Level Response Level
147. orrect rack s are installed In the autosampler when you start the wizard run Help Menu M lt Back gt Close lt o OOOO oOOOOOOOO UN Ku K o A O Biacore 2000 Instrument Handbook 9 Application Wizard 155 3 If Then If Then If Then 2251
148. pplication Template Cancel di This wizard allows vau to program and run customized applications For purposes that are nat covered by the application specific wizards Customized Application Create New Biacore 2000 Instrument Handbook 9 Application Wizard 141 258 Customized Sequence zl 1 Help Back gt Close 20ul min j Settings E Set Temperature w Detection Cancel Detection HRSG ES WANNA va Bv Cycle Help Setting the detection made will alzo set the initial How path Detection e Fc1 FC4 e Fc1 2 FOE e Fc1 2 5 4 e Fc1 2 5 4 Ref 2 1 4 3 Ref 2 1 5 1 4 1 Biacore 2000 Instrument Handbook 142 9 Application Wizard Set Temperature
149. qu C CM Dee De ESSI UNS COMO XE DET SEC C SEC Vase as Da Dac VE 00 6 10 NTA e 4 P Prerun Method s Y e ee e e o o o o o o ooo ooo 27 Prime e 551 53 OUICKINJECT e 39 R Regeneration Scouting Wiazard c sos os eoe oe cem e s m 105 RInse e 5 5 SA e o 4 Sanitize e E CA CS C 4 Uo WO CS DES USU CE ao Sd 54 Specify Flow Rate and Injection Time Wizard e e e ees 101 U Unclogging Ws 55 Undock AR 52 Biacore 2000 Instrument Handbook Biocore 2000 Instrument Hondbook rO ERROA LU EIB PE OREORE A coUv duUlcla 151 89 ms sess CES
150. s s s s s s s s s s s s s s s s s 27 s s s s s s s s s s s s s s s s s s s s s s s s s s s s s 9 18 lt ccc 11 EX s s s s s s s s s s s s s s s s s s s s s s s om s om om om om n n 29 49 EE Ir JU D s s s s s s s s s s s s s s s s s s om om on om n n 24 EE IEEE DEBE D t s s s s s s s s s s s s s s s s e 0305 52 36 84 e s s s o s s o s s 9 9 s 2 2 444499999 39 42 TF AAA s s s s s s s s s s s e s s s s s o s s s s s s s s s s tg n 9 n9 s t t 54 s s s s s sa s s sassa asses 18 19 24 86 87 166 176 uU T e o 56 s s s s s s s s s s s s s c n 3 lt s s s s s s s s s s s s s 9 s 59 9 9 45 Biacore 2000 Instrument Handbook 182 5 e 51 ASIN s s s s s s s s s s s s s s s s s s 9 s s s n n 51 649 905
151. surface performance test Surface Preparation Start am Surface Preparation Surface Preparation Steps Surface Preparation Steps Immobilization pH Scouting Immobilization Regeneration Scouting Surface Performance Test The Immobilization pH scouting wizard guides you through determination af a suitable pH Far immobilizing ligand nn Sensor Chip CBS Help gt Close Biacore 2000 Instrument Hondbook 9 Applicotion Wizard 87 Immobilization pH Scouting Next gt Surface Preparation Buffer Selection 10 mid pH 6 00 Add 10 Acetate pH 5 50 10 mM Acetate pH 5 00 Remove 10 mM Acetate pH 4 50 10 mh Acetate pH 4 00 Prepare ligand In the buffers you have chosen Help lt Back gt Close es 2 00 pH Next gt pH pH pH3 5 pH pH6 4 DRAI d
152. tart Cancel XR OD 1 0 5 1 2r lt 20 60ul min 42 L Start Add OK Add Hew Regeneration Solution 100m HCl Cancel Regeneration Scouting P Ja 1 lt Biacore 2000 Instrument Handbook 110 9 Wizard Regeneration Regeneration Scouting Regeneration Scouting Finish sa Reg Results o _ wam EE 0 3 1D m
153. te Settings M ame Antigen Injection Time Flow Rate 2d Hun Humber of Test Cycles 5 Use Flawcell 3 Help lt Back gt Close pl min 5 Next gt Biacore 2000 Instrument Hondbook 112 9 Wizard 78 ourface Freparation Regeneration Next gt 3 Dissociation in Buffer Single Injection 1 Two Injections 2 2 Biacore 2000 Instrument Handbook 9 Application Wizard 113 Position Vol amp Content R2 Al 360 Antigen THERMO C UE K 0560 E NE K E E OS 1000000 QOOOOOOO
154. tra Cleanup Extra Cleanup Options Predip Heedle Position ly Extra Cleanup 2000 Instrument Hondbook 46 4 Command Queue 1 Edit Delete Edit Insert Command Queue IOl x Fm Edt Run amp QUICKINJECT R2E1 10 QUICKINJECT R2E1 10 QUICKINJECT R2E1 10 FO 10 Cj MIX R2A1 110 EF FLOwPATH 1 2 3 4 QUICKINJECT R2E1 10 Command Queue ug Ju xeguUw ggecmp VO xw e puer cC lt Biacore 2000
155. ues Jb USE D SE wizard Biacore 2000 Instrument Handbook 166 9 Applicqtion Wizard FIFE EJ ESY NJEDRA CEELI 7335 Copo 8 036 BER Sensor Surface Handbook NHS EDC Ie 10mM 5mM 7 77 88 Sensor Surface Handbook amp 88 0 1M Na cyanoborohydride in 0 1M Acetate buffer pH4 10mM Gly HCI pH2 Run Run Application Wizard Zea New Application Wizard Surface Preparation 00 0 3 Binding Analysis Kinetic Analysis Customized Application Create Mew Open Template Cancel qi The Surface preparation application wizard guides wou through the essential steps i
156. wn Bl Unknown Bl LUnknowna3 Bl Unknown4 Unknowns StandardzZ THERMO 006 K KU K sure that the correct rack s are installed In the autosampler when pou start the wizard run Help Menu M lt Back gt Llose THERMD_A j eeeeeccooco00 eeeeceocooo0o0 OOOOOOOODOO OOOOOOOODO SL 0000 O e 72421 260 EL gt i Biacore 2000 Instrument Handbook 164 9 Application Wizard aa 1 Prepare Run E THERMO A right hand rack position Position Volume pl Content A1 Bu standard A B standard A3 Bu standarda Ad B standard A5 B Standards H1 B Unknown 2 B Linknownz B3 Bu Linknovwvn3 B4 B Lnknnwyri B5 Bu Unknowns F3 20 1 Gly HCI pH2 Prime Before Fun Standby Flow After Run _ Hep Menu lt Back Start Close Standby Flow After Prime Before Start es Save Wizard Results As Save In E Demo File name cond Save as Fiesults bir Cancel Sqve
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