AKTA Crystalキューカード
Contents
1. Create the method e Open Method Editor and select Template under File New e Select the Affinity Blank Run 1ml or 5ml template and press OK e Open the Scouting page Use Add to create the number of runs equal to the number of columns that should be equilibrated Select to wash inlet A11 and B1 in the first run Note Even if only one column will be used a scouting run still has to be added e Define column positions for each run Note Default Column Position is set to Position1Bypass which means no column e Save the method Prepare the system e Prepare required buffers see Figure 2 e Place the inlet tubings in the buffers according to Figure 2 e f empty purge the inlet tubings A11 and B1 according to the system pump instructions in the System preparations cue card e Connect the affinity columns at defined column positions Start the method The method takes approximately 10 minutes column to run All B1 Affinity Affinity binding elution and wash buffer buffer 100 ml 150 ml Fig 2 Buffers required to run the Affinity Blank Run template lon exchange Desalting and Gel filtration columns Equilibrate columns Purpose To remove EtOH and equilibrate columns with buffer Prepare the system e Prepare sufficient volume of the required buffer see Table 1 e Place the inlet tubing e g A12 in the equilibration buffer and fill the inlet according to the system pump instruct2. sample 5 HiTrap HiTrap sample 3 sample 4 Fig 2 Column connections at column valves V2 and V3 Prepare and load samples e Prepare your samples and clarify them using centri fugation and or filtration through a 0 45 um filter e Prepare the sample pump according to the System preparations cue card Gently transfer each sample inlet tubing into each sample according to Figure 3 Make sure no air enters the tubing S4 S5 S8 W IYYYY B Affinity binding and wash buffer 0 51 Fig 3 Sample placement at the sample valve V5 4 Start the method e Start your prepared method In the start protocol e Key in sample ID for the proteins on the variable page Each chromatogram will be named after the sample ID e Start your automated multi step purification 5 Evaluate results e How to view the results in an easy way and how to calculate protein concentrations is described in AKTA 3D plus Kit User Manual and on the Results and evaluation cue card Cue card 11 0014 58 AB 7 Protocol C Affinity Gel filtration This cue card describes how to run this specific purification protocol Before starting a purification protocol make sure to prepare system and columns according to the instructions on the system preparations buffer suggestions and column preparations cue cards 1 Prepare your UNICORN method e Open the method wizard and select purification protocol C Affinity Step Gel Fi
3. Any HiTrap GSTrap FF or GSTrap HP can be used 2 Prepare your system before a run Prepare buffers required for this protein purification protocol according to the Buffer suggestions and column preparations cue card Place buffer inlets as shown in Figure 1 and make sure your system is prepared as described in the System preparations cue card 10 Cue card 11 0014 58 AB Connect selected columns as shown in Figure 2 and prepare them according to the Buffer suggestions and column preparations cue card Load the fraction collector with four 96 well micro plates 2 ml You will also need to load the fraction collector with 50 ml fraction tubes to collect the second wash of the affinity column For more details see AKTA 3D plus Kit User Manual Affinity Desalting Affinity binding buffer elution and wash 161 buffer buffer 0 51 1 01 Fig 1 Buffer inlets and maximum volumes needed see AKTA 3D plus Kit User Manual for details HiPrep Desalting sample 1 4 N Bypass HiTrap HiTrap sample 1 yg y sample 6 HiTrap t Mis sample 2 HiTrap sample 5 HiTrap HiTrap sample 3 sample 4 Fig 2 Column connections at column valves V2 and V3 Protocol D Affinity Desalting continued 3 Prepare and load samples 4 Start the method e Prepare your samples and clarify them using e Start your prepared method centrifugation and or filtration through a 0 45 um filter e Prepare the sample pump a
4. and maximum volumes needed see AKTA 3D plus Kit User Manual for details Cue card 11 0014 58 AB Bypass HiTrap sample 1 ag HiTrap y sample 6 dig HiTrap sample 5 HiTrap sample 2 HiTrap sample 3 HiTrap sample 4 Fig 2 Column connections at column valves V2 and V3 Prepare and load samples e Prepare your samples and clarify them using centrifugation and or filtration through a 0 45 um filter e Prepare the sample pump according to the System preparations cue card Gently transfer each sample inlet tubing into each sample according to Figure 3 Make sure no air enters the tubing 5 S1 S3 S4 S5 S6 iL VV Fig 3 Sample placement at the sample valve V5 Affinity binding and wash buffer 0 51 4 Start the method e Start your prepared method In the start protocol e Key in sample ID for the proteins on the variable page Each chromatogram will be named after the sample ID 5 Evaluate results e How to view the results in an easy way and how to calculate protein concentrations is described in AKTA 3D plus Kit User Manual and on the Results and evaluation cue card Protocol B Affinity gradient elution This cue card describes how to run this specific purification protocol Before starting a purification protocol make sure to prepare system and columns according to the instructions on the system preparations buffer suggestions and column preparatio
5. e Prepare buffers required for this protein purification protocol according to the Buffer suggestions and column preparations cue card e Place buffer inlets as shown in Figure 1 and make sure your system is prepared as described in the System preparations cue card 12 Cue card 11 0014 58 AB i RESOURCE Q HiTrap sample 1 4 sample 1 ay HiTrap sample 2 HiTrap HiTrap sample 3 sample 4 Fig 2 Column connections at column valves V2 and V3 Protocol E Affinity Desalting lon Exchange continued 3 Prepare and load samples 4 Start the method e Prepare your samples and clarify them using e Start your prepared method centrifugation and or filtration through a 0 45 um filter e Prepare the sample pump according to the System In the start protocol e Key in sample ID for the proteins on the variable page Each chromatogram will be named after the preparations cue card Gently transfer each sample sample ID inlet tubing into each sample according to Figure 3 Start your automated multi step purification Make sure no air enters the tubing 5 Evaluate results Lol G e How to view the results in an easy way and how to calculate protein concentrations is described in AKTA 3D plus Kit User Manual and on the Results and evaluation cue card l Affinity binding and wash buffer 0 51 S2 S3 S4 8 Fig 3 Sample placement at the sample valve V5 Cue card 11 0014 58 AB 13 S
6. well plates 2 ml or 120 tubes 10 ml depending on your choice of gel filtration column see Table 1 You will also need to fill the fraction collector with 50 ml fraction tubes to collect the second wash of the affinity column one tube per sample is needed a A f All Al2 B1 Affinity Gel filtration Affinity binding buffer elution and wash 271 buffer buffer 0 51 1 21 Fig 1 Buffer inlets and maximum volumes needed see KTA 3D plus Kit User Manual for details Ld HiLoad Superdex sample 1 6 Bypass HiTrap K JJ HiTrap sample 1 ag Ww sample 6 Ar A HiTrap sample 2 N A HiTrap sample 5 HiTrap HiTrap sample 3 sample 4 Fig 2 Column connections at column valves V2 and V3 Protocol C Affinity Gel filtration continued 3 Prepare and load samples 4 Start the method e Prepare your samples and clarify them using e Start your prepared method centrifugation and or filtration through a 0 45 um filter e Prepare the sample pump according to the System In the start protocol e Key in sample ID for the proteins on the variable page Each chromatogram will be named after the preparations cue card Gently transfer each sample sample ID inlet tubing into each sample according to Figure 3 Start your automated multi step purification Make sure no air enters the tubing 5 Evaluate results e How to view the results in an easy way and how to calculate protein concentrati
7. GE Healthcare Cue card 11 0014 58 AB Automated multi step protein purification System preparations Buffer suggestions Column preparations Purification protocols System maintenance Column cleaning Results and Evaluation KTA 3D plus Kit System preparations This cue card describes general system preparations To ensure the best results use high purity water and chemicals It is also recommended to filter the solvents through a 0 45 um filter Available protocols Table 1 Steps in available protocols Protocol Included steps A Affinity AC step B Affinity AC gradient Affinity AC Gel filtration GF D Affinity AC Desalting DS E Affinity AC Desalting DS lon Exchange IEX Column positions Table 2 Column positions for protocols A E Column Protocol position A B iG D E 2 ACI1 AC 1 AC 1 AC 1 AC 1 3 AC 2 AC 2 ACI2 AC 2 AC 2 4 AC 3 Ac 3 ACI3 AC 3 AC 3 5 AC 4 ACA ACI4 AC 4 ACI4 6 AC 5 Ac 5 ACIS AC 5 7 AC 6 AC 6 AC 6 IEX 8 GF DS DS System Pump Fill buffer inlet tubings e Prepare required buffers according to the chosen purification protocol Add extra volume for system and column preparation e Place the inlet tubings in the buffers according to Table 3 e If empty purge the buffer inlet tubings manually according to P 900 User manual 2 Cue card 11 0014 58 AB Table 3 Buffer positions for pr
8. Valve V8 and replace it with a 10 ml loop Note If the loop is empty fill it with buffer before the purification Fill it manually with a syringe or use the template System and Loop wash e Create a method and check Show details in the Run Setup dialog e Change volumes for the variables displayed in the table below Table 4 Variables to change value of if using a 10 ml loop instead of the default 5 ml loop in Loop_1 Valve V8 Change values for all protocols that are used Default New Block Variable ml ml Wash_Loop1_Che Wash_Volume_Loop _Che 25 50 or Wash_Loop1_GST Wash_Volume_Loop _GST 25 50 Hold_Until_Peak_Less_Than Max_CollectionVolume 3 5 6 5 Inject_Collected_peak Loop_1_Volume 4 7 Delay_inject_selected_peak InjectionDelay 5 10 1 The default values are for 5 ml HiTrap columns The new values are examples that are useable if using a HiPrep Desalting or a HiLoad Superdex 26 60 in the second step 3 Depending on the type of column used Cue card 11 0014 58 AB 3 Buffer suggestions and column preparations This cue card describes how to prepare different columns before running a protocol To ensure the best results use high purity water and chemicals It is also recommended to filter the solvents through a 0 45 um filter Buffer suggestions AC buffer suggestions for His tagged proteins If performing suggested buffer binding using 50 mM Tris HCl pH 7 5 0 5 M NaCl HisTr
9. ap 20 40 mM imidazole1 binding using 50 mM Tris HCl pH 7 5 0 5 M NaCl HiTrap Chelating 5 40 mM imidazole1 elution 50 mM Tris HCl pH 7 5 0 5 M NaCl 500 mM imidazole 1 The imidazole concentration is protein dependent AC buffer suggestions for GST tagged proteins If performing suggested buffer binding using 50 mM Tris HCl pH 7 5 150 mM NaCl GSTrap HP or FF 1 mM DIT elution 50 mM Tris HCl 0 15 M NaCl 1 mM DTT 10 mM reduced gluthathione pH 8 DS buffer suggestions If preparing for suggested buffer AIEX 50 mM Tris HCl pH 8 0 CIEX 20 mM MES pH 6 0 protein storage include e g 10 glycerol in a suitable buffer e g 50 mM Tris HCl pH 7 5 150 mM NaCl IEX buffer suggestions If for example suggested buffer depends on the pl of the protein binding to AIEX 50 mM Tris HCl pH 8 0 binding to CIEX 20 mM MES pH 6 0 eluting from AIEX 50 mM Tris HCl pH 8 0 1 M NaCl eluting from CIEX 20 mM MES pH 6 0 1 M NaCl GF buffer suggestions If preparing for suggested buffer further studies a suitable buffer e g 50 mM Tris HCl pH 7 5 150 mM NaCl include e g 10 glycerol in a suitable buffer e g 50 mM Tris HCl pH 7 5 150 mM NaCl protein storage 4 Cuecard 11 0014 58 AB Column preparations Affinity Columns Metal ion charging Purpose To charge new or stripped HiTrap Chelating HP or HisTrap columns with metal ions e g Ni Co or Cu Several columns can be pr
10. atography AkTAexplorer100 with Pump P 960 and UNICORN 5 01 or higher and RESOURCE Q 1lxim 7 1177 0 Fraction collector Frac 950 with Rack A and Rack C Rack A is the standard RESOURCE Q 1x6m 7 1179 0 rack supplied with Fraction collector Frac 950 are needed to use AKTA 3D plus Kit AKTAexplorer 100 upgrades can be ordered from your local LabcrewTM RESOURCE S 1x1lm 7 1178 0 representative of GE Healthcare A representative of GE Healthcare is required to RESOURCE S 1x6m 7 1180 0 install AKTA 3D plus Kit HiTrap Q HP 5xim 7 1153 0 GE Healthcare recommends Greiner PP Masterblock 2 ml 96 well 780270 HiTrap Q HP 5x5m 7 1154 0 18 Cue card 11 0014 58 AB Trouble shooting and useful hints can be found in the User Manual 11 0014 57 AA www gelifesciences com GE Healthcare Bio Sciences AB Bj rkgatan 30 751 84 Uppsala Sweden GE imagination at work and GE monogram are trademarks of General Electric Company AKTA DropDesign GSTrap HiLoad HiPrep HisTrap HiTrap Labcrew Mono Q Mono S RESOURCE Superdex and UNICORN are trademarks of GE Healthcare companies All third party trademarks are the property of their respective owners 2004 2007 General Electric Company All rights reserved First published Oct 2004 All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them A copy of these terms and conditions is available on reques
11. ax 47 815 65 666 e Portugal Tel 21 417 7035 Fax 21 417 3184 e Russia amp other C I S amp N I S Tel 7 495 956 5177 Fax 7 495 956 5176 e Spain Tel 902 11 72 65 Fax 935 94 49 65 e Sweden Tel 018 612 1900 Fax 018 612 1910 e Switzerland Tel 0848 8028 10 Fax 0848 8028 11 e UK Tel 0800 515 313 Fax 0800 616 927 e USA Tel 1 800 526 3593 Fax 1 877 295 8102 imagination at work 11 0014 58 AB 05 2007 EE Elanders sterv la 2007
12. cation protocol make sure to prepare system and columns according to the instructions on the system preparations buffer suggestions and column preparations cue cards 1 Prepare your UNICORN method Open the method wizard and select purification protocol A Affinity Step ark the number of samples 1 to 6 to be purified Press Next Choose columns ndicate your running condition room temperature or cold room Press Finish to obtain the purification method t is possible to change default values in the method if needed Check the Show details box For more details see AKTA 3D plus Kit User Manual Save your method 2 Prepare your system before a run 6 Prepare buffers required for this protein purification protocol according to the Buffer suggestions and column preparations cue card Place buffer inlets as shown in Figure 1 and make sure your system is prepared as described in the System preparations cue card Connect selected columns as shown in Figure 2 and prepare them according to the Buffer suggestions and column preparations cue card Load the fraction collector with four 96 well micro plates 2 ml You will also need to load the fraction collector with 50 ml fraction tubes to collect the second wash of the affinity column For more details see AKTA 3D plus Kit User Manual f All B1 Affinity Affinity binding elution and wash buffer buffer 0 51 121 Fig 1 Buffer inlets
13. ccording to the System In the start protocol e Key in sample ID for the proteins on the variable page Each chromatogram will be named after the preparations cue card Gently transfer each sample sample ID inlet tubing into each sample according to Figure 3 Start your automated multi step purification Make sure no air enters the tubing 5 Evaluate results e How to view the results in an easy way and how to calculate protein concentrations is described in f KTA 3D plus Kit User Manual and on the Results S S8 95 34 58 Se ii and evaluation cue card i fH J y WY VW J Affinity binding and wash buffer 0 51 Fig 3 Sample placement at the sample valve V5 Cue card 11 0014 58AB 11 Protocol E Affinity Desalting lon Exchange This cue card describes how to run this specific purification protocol Before starting a purification protocol make sure to prepare system and columns according to the instructions on the system preparations buffer suggestions and column preparation cue cards 1 Prepare your UNICORN method e Open the method wizard S and select purification protocol E Affinity step desalting IEX e ark the number of samples 1 to 4 to be purified e Press Next e Choose columns See Table 1 for recommendations e Indicate your running condition room temperature or cold room e Press Finish to obtain the purification method e It is possible to cha
14. curves to be displayed commonly 1 4 6 11 and 22 If the Clear function is used also remember to scroll down and select curve 22 e Press OK to confirm your selections Note If you have entered the sample ID on the variable page the sample ID will be shown in the chromatogram heading 3 Pool fractions and adjust pooling e If required maximize the sample window Bij e Zoom iin on the relevant fractions e Choose Operations Pool to pool the fractions The pooled fractions are listed in a table below the chromatogram and the pooled peaks are numbered sequentially in the chromatogram Only adjacent fractions will be pooled The fraction numbers for each pool are listed in the table as a range in retention order e g A6 A7 etc e f necessary adjust pooling Vol Pool Ara Ext Coeff Result and evaluation continued 4 Determine protein concentration and amount Pooling protocol e Enter the real cell lengt of the UV cell in the Path Lenght cm field Note If the real cell lenght has been set direcly in Monitor UV 900 enter the nominal cell lenght 0 2 cm instead e Enter the extinction coefficient manually by marking a pool and then typing the value in the extinction coefficient field Ce rent oso Har wee The concentration is given in mg ml or M and amount is given in mg or mmole depending on the 5 Print or save the Pooling protocol coefficient used The Pooli
15. epared automatically Create the method e Open Method Editor and select Template under File New e Select the Metal lon Charging 1ml or 5ml template and press OK e Open the Scouting page Use Add to create the number of runs equal to the number of columns that should be charged Note Even if only one column will be used a scouting run still has to be added e Define column positions for each run Note Default Column Position is set to Position1Bypass which means no column e Save the method Prepare the system e Prepare required solutions see Figure 1 e Connect the chelating columns at defined column positions e Place the inlet tubings in the solutions according to Figure 1 e Fill the inlet tubings S1 S2 and S8 and purge the sample pump according to the instructions in the System preparations cue card Start the method The method takes approximately 15 minutes column to run G 7 ie L i i S1 S2 S8 Water 0 7 Metal Affinity solution binding and 100 ml wash buffer e g 0 1M 0 51 NiSO Fig 1 Solutions required to run the Metal lon Charging template Buffer suggestions and column preparations continued Affinity blank run Purpose Prior to a first time usage of an affinity column GSTrap FF GSTrap HP or a newly metal charged HiTrap Chelating HP or HisTrap it is recommended to run a blank run This ensures a well conditioned and equilibrated column ready for chromatography
16. he purge tubing to the same port e Make sure the injection valve V1 is set to LOAD e Draw buffer with the syringe until liquid enters the syringe e Switch the sample valve V5 to the next sample inlet tubing to be filled e Repeat the 2 steps above marked with for the remaining sample inlet tubings e Disconnect the purge tubing e Connect the original connector to the port B Remove small amounts of air from the sample tubing Purpose To remove small amounts of air in the sample flow path by running the sample pump manually e Put all the sample inlet tubings into affinity binding buffer e Open System Control and select Manual Flowpath e Choose the instruction SampleValve select the appropriate position and press Execute e Select Pump and choose SampleFlow e Enter an appropriate flow rate e g 1 ml min and press Execute e When the selected inlet is filled with solution change sample valve position and fill all sample inlets to be used the same way e When all sample inlets have been filled set sample valve position to S8 and run the sample pump at flow rate 40 ml min for 1 min to make sure that no air remains in the sample flow path Change loop in Loop_1 Valve V8 By default the 5 ml loop is connected to Loop_1 Valve V8 If running AC DS or AC GF with large columns and large protein amounts it is possible to increase the yield by using a 10 ml loop instead e Remove the 5 ml loop from the Loop_1
17. hould be lower than the pressure limit RT room temperature 3 CR cold room System maintenance and column cleaning continued Metal lon Stripping chelating columns Purpose To remove metal ions before regenerating the HiTrap HP and HisTrap columns To recharge the column follow the instructions given on the Buffer suggestions and column preparations cue card Note Always remove metal ions before or right after storing the HiTrap Chelating and HisTrap columns in EtOH Create the method e Open Method Editor and select Template under File New e Select the Metal lon Stripping 1ml or 5ml template and press OK e Open the Scouting page Use Add to create the number of runs equal to the number of columns that should be stripped Note Even if only one column will be used a scouting run still has to be added e Define Column Position for each run e Save the method Prepare the system e Prepare required solutions see Figure 1 e Connect the columns at defined column positions e Place the inlet tubings in the solutions according to Figure 1 Fill the inlet tubings S1 S3 and S8 and purge the sample pump according to instructions in the System preparations cue card Start the method The method takes approximately 20 minutes column to run AL L _ff S1 S3 S8 Water 0 5 I Metal Affinity stripping binding and solution wash buffer e g 50 mM 0 51 EDTA 100 ml Fig 1 Solutions re
18. ions given in the System preparation cue card e Connect the columns Manual run Equilibration of columns is performed manually from System Control Note After each selection below always press Execute e Select Alarm_Pressure in Manual Alarms amp Mon and key in HighAlarm see Table 1 e Select BufferValveA1 A12 in Manual Flowpath e Select appropriate ColumnPosition in Manual Flowpath e Select Flow in Manual Pump key in appropriate flow see Table 1 e Select End_timer in Manual Other and choose Acc volume key in appropriate Timeout volume see Table 1 e When the first column is ready repeat for the next column to be equilibrated Note To make automated methods see UNICORN User Manual Table 1 Recommended HighAlarms Flow rates and Equilibration volumes for different columns when running AKTA 3D plus Kit including a 0 2 MPa flow restrictor Timeout Flow rate Flow rate Equilibration High Alarm removing EtOH equilibration volume Column MPa ml min ml min ml HiLoad 16 60 0 5 0 5 1 0 360 HiLoad 26 60 0 5 13 25 954 HiPrep 26 10 0 35 7 5 15 0 159 HiTrap 1ml 0 5 0 5 1 0 5 HiTrap 5ml 0 5 25 5 0 25 RESOURCE 1ml 15 2 0 40 5 RESOURCE 6ml 15 3 0 6 0 30 Mono Q 5 50 GL 4 0 10 2 0 Mono S 5 50 GL 4 0 1 0 2 0 Cue card 11 0014 58 AB 5 Protocol A Affinity step elution This cue card describes how to run this specific purification protocol Before starting a purifi
19. ltration e ark the number of samples 1 to 6 to be purified e Press Next e Choose columns See Table 1 for recommendations e Indicate your running condition room temperature or cold room e Press Finish to obtain the purification method e It is possible to change default values in the method if needed Check the Show details box For more details see AKTA 3D plus Kit User Manual e Save your method Table 1 Recommended column combinations Goal __First column _ Second column Frac 950 Upto HiTrap 1ml HiLoad 16 60 Superdex 75 Rack C with i 10 mg prep grade micro titre protein or plates and HiLoad 16 60 Superdex 200 50 ml tubes prep grade Upto HiTrap 5 ml HiLoad 26 60 Superdex 75 Rack A with 50 mg prep grade 10 and 50 ml protein or tubes HiLoad 26 60 Superdex 200 prep grade 1 Either of HiTrap Chelating HP HisTrap Any HiTrap GSTrap FF or GSTrap HP can be used 2 Prepare your system before a run e Prepare buffers required for this protein purification protocol according to the Buffer suggestions and column preparations cue card e Place buffer inlets as shown in Figure 1 and make sure your system is prepared as described in the System preparations cue card 8 Cue card 11 0014 58 AB e Connect selected columns as shown in Figure 2 and prepare them according to the Buffer suggestions and column preparations cue card e Fill the fraction collector with four 96
20. ng protocol can be used as a help when making e Click the Add to Pooling Protocol button to add the the physical pooling of the purified samples from the adjusted pools to the Pooling protocol microplate e Repeat the procedure for other chromatograms from the same or other result files a Ep pringthe Pooling protocol x Click the View Pooling Protocol button Click the Print button to print the protocol on the default printer e To save the Pooling protocol as a file Click Export and save the protocol in one of the following formats text txt Excel xls HTML htm Cue card 11 0014 58 AB 17 Ordering information Product Pack Size Code No Product Pack Size Code No KTA 3D plus Kit with HiTrap SP HP 5x1m 7 1151 0 software valve INV 907 HiTrap SP HP 5x5m 7 1152 0 air sensor 915 N flow HiTrap Q FF 5x1m 7 5053 0 restrictor FR 902 tubings ne HiTrap Q FF 5x5m 7 5156 0 sample loops fittings A z HiTrap SP FF 5x1m 7 5054 0 instruction manual and eae cards 11 0014 53 HiTrap SP FF 5x5m 7 5157 0 AKTAexplorer 100 please contact HiTrap DEAE FF 5x1m 7 5055 0 your local sales HiTrap DEAE FF 5x5m 7 5154 0 representative HiTrap CM FF 5x1m 7 5056 0 of GE Healthcare HiTrap CM FF 5x5m 7 5155 0 Fraction collector Frac 950 HiTrap ANX FF high sub 5x1m 7 5162 0 complete with Rack A 18 and HiTrap ANX FF high sub 5x5m 7 5163 0 30 mm tubes 8 6083 00 i HiT
21. nge default values in the method if needed Check the Show details box For more details see AKTA 3D plus Kit User Manual e Save your method Connect selected co umns as in Figure 2 and prepare them according to the Buffer suggestions and column preparations cue card Load the fraction co lector with four 96 well microplates 2 ml You will also need to load the fraction collector wit the second wash of flowthrough Two tu h 50 ml fraction tubes to collect he affinity column and the IEX bes per sample are needed For more details see AKTA 3D plus Kit User Manual f A11 A12 B1 B2 Affinity Desalting Affinity IEX elution binding and IEX elution buffer and wash binding buffer 0 51 buffer buffer 0 51 1 01 161 Table 1 Recommended column combinations Goal First column Second column Third column Frac 950 Fig 1 Buffer inlets and maximum volumes needed see AKTA 3D plus Kit User Manual for details HiPrep Desalting sample 1 4 Bypass Upto HiTrapt 1ml 2x HiTrap 1 50r6ml Rack C with 10 mg Desalting IEX micro titre protein or plates and HiPrep 50 ml tubes Desalting Upto HiTrapt 5ml HiPrep Desalting 5 or 6 ml Rack C with 50 mg IEX micro titre protein plates and 50 ml tubes 1 Either of HiTrap Chelating HP HisTrap Any HiTrap GSTrap FF or GSTrap HP can be used AIEX for proteins with low pl CIEX for proteins with high pl 2 Prepare your system before a run
22. ns cue cards 1 Prepare your UNICORN method e Open the method wizard and select purification protocol B Affinity Gradient e ark the number of samples 1 to 5 to be purified e Press Next e Choose columns e Indicate your running condition room temperature or cold room e Press Finish to obtain the purification method e It is possible to change default values in the method if needed Check the Show details box For more details see AKTA 3D plus Kit User Manual e Save your method 2 Prepare your system before a run e Prepare buffers required for this protein purification protocol according to the Buffer suggestions and column preparations cue card e Place buffer inlets as shown in Figure 1 and make sure your system is prepared as described in the System preparations cue card e Connect selected columns as shown in Figure 2 and prepare them according to the Buffer suggestions and column preparations cue card e Load the fraction collector with four 96 well microplates 2 ml You will also need to load the fraction collector with 50 ml fraction tubes to collect the second wash of the affinity column For more details see AKTA 3D plus Kit User Manual All B1 Affinity Affinity binding elution and wash buffer buffer 0 51 121 Fig 1 Buffer inlets and maximum volumes needed see AKTA 3D plus Kit User Manual for details Bypass HiTrap sample 1 y HiTrap i iis sample 2 me HiTrap
23. ons is described in LI x A eS H KTA 3D plus Kit User Manual and on the Results SL 52 193 34 35 6 8 and evaluation cue card yY YV i UR Affinity binding and wash buffer Fig 3 Sample placement at the sample valve V5 Cue card 11 0014 58 AB 9 Protocol D Affinity Desalting This cue card describes how to run this specific purification protocol Before starting a purification protocol make sure to prepare system and columns according to the instructions on the system preparations buffer suggestions and column preparations cue cards 1 Prepare your UNICORN method Open the method wizard and select purification protocol D Affinity Step Desalting ark the number of samples 1 to 6 to be purified Press Next Choose columns See Table 1 for recommendations ndicate your running condition room temperature or cold room Press Finish to obtain the purification method t is possible to change default values in the method if needed Check the Show details box For more details see AKTA 3D plus Kit User Manual Save your method Table 1 Recommended column combinations Goal First column Second column _Frac 950 Upto HiTrapt 1 m 2 x HiTrap DS Rack C with micro titre 10 mg or plates and 50 ml tubes protein HiPrep DS Upto HiTrap 5 m 2 x HiTrap DS Rack C with micro titre 50 mg or plates and 50 ml tubes protein HiPrep DS 1 Either of HiTrap Chelating HP HisTrap
24. otocols A E Buffer Protocol inlet A B c D E A11 AC binding AC binding AC binding AC binding AC binding A12 GF DS IEX binding B1 AC elution AC elution AC elution AC elution AC elution B2 IEX elution The IEX binding buffer is also used for desalting Wash system pump e Open System Control and select Manual Pump e Mark the instruction Pump Wash Explorer and select appropriate pump inlets e g A11 B1 B2 Each pump wash requires approximately 50 ml buffer e Press Execute to start the wash e Press End when the wash is completed Note If more than one InletA1 needs to be washed e g A11 and A12 the pump wash must be repeated Sample Pump Presence of air bubbles in the pump and or sample flow path will affect the flow rate Air must therefore be remo ved according to A or B below before starting the run If the sample pump is empty and or large amounts of the tubing is filled with air proceed as in A below To remove small amounts of air proceed as in B on next page A Remove large amounts of air using a purge tubing Purpose To remove large amounts of air in the sample flow path by using a purge tubing and syringe e Put all the sample inlet tubings into affinity binding buffer e Set the sample valve V5 to any of the chosen sample inlet ports e Disconnect the connector fitted to injection valve V1 port 4 System preparations continued e Connect t
25. quired for the Metal lon Stripping template Cue card 11 0014 58 AB 15 Results and evaluation This cue card describes how to open results and determine protein concentration and amount 1 Find and open the result files e Use the Recent Runs or the Find tab in the Evaluation module to locate the result file e Click to expand the list for the result file A result file sli consists of Sample loading chromatogram 11 below Purification chromatograms for each sample e Double click a sample ID to open that specific sample chromatogram e Double click a result file to open all included chromatograms The first chromatogram shows the loading of all samples onto the affinity columns Each one of the remaining chromatograms shows the purification of one individual sample File Edit Yiew Integrate Operation nesal Sl a oO fe Recent Runs Files Find Recent Results AC DS Ui AC DS IEX 001 7 Sample1 Sample2 Note To locate a specific sample click the Find tab enter sample ID and click Quick Find Recent Runs Files Find Result file name Value of variable Sample_ID p450 a Quick Find Find E U System1001 o Home fe si System1002 o Home 16 Cue card 11 0014 58 AB 2 Select curves to display e Right click on the chromatogram and select Properties e Inthe curve selection table select
26. rap Q XL 5x1lm 7 5158 0 Rack C complete with bowl pi A eE O for 96 well microtitre plates tapo worm 7 and 30 mm tubes 8 6083 13 HiT rap SP AL Sam Polen Supported Columns HiTrap SP XL 5x5m 7 5161 0 Affinity chromatography ono Q 5 50 GL xim 7 5166 0 HiTrap Chelating HP 5x1m 7 0408 0 ono 5 50 GL xam 7 5168 0 HiTrap Chelating HP 1x5m 7 0409 0 P 2 Related products and literature HisTrap HP 5x1m 7 5247 0 HiST HP 5x5 7 5248 02 gt 2 AOR eau KTA 3D plus Kit User Manual 1 0014 57 GSTrap FF 5x1m 7 5130 0 Test Kit 280 nm 2 mm cell 8 1129 63 GSTrap FF 2x1m 7 5130 02 KTA 3D plus Kit Miniposter 1 0025 34 GSTrap FF 1x5m 7 5131 0 HiTrap Column Guide 8 1129 81 GSTrap HP 5x1m 7 5281 0 7 ee He e PERN KTAexplorer chromatography HR sere 7 systems Data File 8 1124 09 Gel filtration Fraction collector Frac 950 HiLoad 16 60 Superdex 75 Data File 8 1153 57 prep grade 1x 120 ml 7 1068 0 Affinity Chromatography HiLoad 16 60 Superdex 200 Handboo 8 1022 29 rep grade 1x 120 ml 7 1069 0 PERA Gel Filtration Handbook 8 1022 18 HiLoad 26 60 S dex 75 ips i aca ENTE A lon Exchange Chromatography prep grade x m 1 Handbool 8 1114 21 HiLoad 26 60 S dex 200 x P ma GU Superdex Recombinant Protein Handbook 8 1142 75 prep grade 1x318 ml 7 1071 0 GST Gene Fusion System Buffer exchange Handbool 8 1157 58 HiPrep 26 10 Desalting 1x 53 ml 7 5087 0 Purifying Challenging Proteins HiTrap Desalting 5x5m 7 1408 0 Handbool 28 9095 31 lon exchange chrom
27. rd 11 0014 58 AB Create the method e Open Method Editor and select Template under File New e Select the Column CIP template and press OK e Open the Scouting page Use Add to create the number of runs equal to the number of columns that should be cleaned Note Even if only one column will be used a scouting run still has to be added e Define Column volume Pressure limit Regulation pressure Flow rate and Column position for each run See table 1 for recommendations e Select the buffer inlets flow rates and volumes for all solutions that will be used Up to 10 steps per column can be performed e Save the method Prepare the system e Prepare a sufficient volume of each required solution e Connect the columns at defined column positions e Place the inlet tubings in appropriate solutions e Fill the inlet tubings according to System Pump instructions in the System preparations cue card Start the method Table 1 CIP values to enter on the scouting page if using FR 902 flow restrictor Column Pressure Regulation Flow Flow Column tal wea nein ein HiTrap 1 ml 0 96 0 5 0 45 1 0 0 8 HiTrap 5 ml 5 0 0 5 0 45 5 0 40 HiPrep DS 53 0 35 0 3 10 8 0 RESOURCE 1m 0 97 15 13 4 0 3 2 RESOURCE6ml 6 0 15 13 6 0 48 onoQ5 50GL_ 0 98 40 3 6 2 0 1 6 onoS5 50GL 0 98 40 3 6 2 0 1 6 HiLoad 16 60 121 0 5 0 45 1 0 0 8 HiLoad 26 60 319 0 5 0 45 2 5 2 0 1 Target pressure used by the pressure flow regulation S
28. t Contact your local GE Healthcare representative for the most current information GE Healthcare Europe GmbH Munzinger Strasse 5 D 79111 Freiburg Germany GE Healthcare UK Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK GE Healthcare Bio Sciences Corp 800 Centennial Avenue PO Box 1327 Piscataway NJ 08855 1327 USA GE Healthcare Bio Sciences KK Sanken Bldg 3 25 1 Hyakunincho Shinjuku ku Tokyo 169 0073 Japan Asia Pacific Tel 85 65 62751830 Fax 85 65 62751829 Australasia Tel 61 2 8820 8299 Fax 61 2 8820 8200 e Austria Tel 01 57606 1613 Fax 01 57606 1614 e Belgium Tel 0800 73 890 Fax 02 416 8206 Canada Tel 1 800 463 5800 Fax 1 800 567 1008 e Central East amp South East Europe Tel 43 1 972 720 Fax 43 1972 722 750 e Denmark Tel 45 70 25 24 50 Fax 45 45 16 2424 e Eire Tel 1 800 709992 Fax 44 1494 542010 e Finland amp Baltics Tel 358 9 512 3940 Fax 358 9 512 39439 e France Tel 01 69 35 67 00 Fax 01 69 41 98 77 Germany Tel 0800 9080 711 Fax 0800 9080 712 Greater China Tel 852 2100 6300 Fax 852 2100 6338 e Italy Tel 02 26001 320 Fax 02 26001 399 e Japan Tel 81 3 5331 9336 Fax 81 3 5331 9370 e Korea Tel 82 2 6201 3700 Fax 82 2 6201 3803 e Latin America Tel 55 11 3933 7300 Fax 55 11 3933 7304 Middle East amp Africa Tel 30 210 96 00 687 Fax 30 210 96 00 693 e Netherlands Tel 0800 82 82 82 1 Fax 0800 82 82 82 4 e Norway Tel 47 815 65 777 F
29. ystem maintenance and column cleaning This cue card describes how to clean your system and columns after performed protein purification To ensure the best results use high purity water and chemicals It is also recommended to filter buffers through a 0 45 um filter System and Loop Wash Purpose To wash all used tubings within the system including the loops Create the method Open Method Editor and select Template under File New Select the System and Loop Wash template and press OK Select up to two buffer inlets to be washed at the same time as the system e g A11 and B1 Note If more than one InletA1 needs washing e g A11 and A12 we recommend to clean these inlets according to the System Pump instructions in the System preparations cue card before performing the System Wash Sample inlet tubings must be washed separately according to the Sample Pump instructions in the System preparations cue card Save the method Prepare the system Prepare a sufficient volume 0 4 of the wash solution Place the inlet tubings in the wash solution Start the method The method takes approximately 10 minutes to run Column CIP cleaning in place Purpose To clean contaminated columns For column cleaning procedures and column storage instructions please refer to the instructions supplied with each column or to our homepage www gelifesciences com chromatography 14 Cue ca
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