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ÄKTAprime plus - GE Healthcare Life Sciences

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1. Y Set Drop Sync Active yes Note Drop synchronization can only be used at flow rates lt 3 ml min Cleaning the sample loop and connecting the column Mount a sample loop between port 2 and port 6 that is large enough to hold your sample Clean the loop and the injector fill port Inject with a syringe 5 times the loop volume of water or binding buffer through the injection fill port c Connect the column between port 1 on the injection valve 7 port valve and the UV flow cell 1 16 Male 1 16 Male HiTrap o UV monitor HiPrep 1 16 Male 8 Filling the buffer inlet tubing Prepare the buffers Make sure that correct inlet tubing is put into correct buffer If using an application template Itis not necessary to perform the wash procedure because this is included in the method If using a method template Pre fill all inlet tubings with buffer Select the application template System Wash Method select the inlets to be washed and press OK The system is now prepared Start the method 9 Storage of the system a b c Clean the loop and the injector fill port e Inject with a syringe 5 times the loop volume of water through the injection fill port e Repeat this step with 20 ethanol Flush the system flow paths e Put all used inlet tubings in water e Select the application template System Wash Method select the inlets to be washed and pres
2. Re equilibration 11 10 20 17 5 Min Total separation time 63 min sample application time 5 Typical result Ordering information Sample Protein mix containing transferrin ovalbumin and Product Quantity Code No B lactoglobulin in start buffer A Column HiTrap Q HP 1ml HiTrap Q HP 5x1m 17 1153 01 Start buffer A1 20 mM Tris HCl pH 8 0 na 4 Elution buffer B 20 mM Tris HCl 1 0 M NaCl pH 8 0 HiTrap Q FF 5x 1m 7 5053 01 AUoa0 nm B HiTrap DEAE FF 5x1m 7 5055 01 UV 280 nm HiTrap ANX FF high sub 5x1m 17 5162 01 0 20 Programmed B 80 HiTrap Q XL 5x1m 17 5158 01 HiTrap Capto Q 5x1m 11 0013 02 0 15 m 60 HiTrap Capto DEAE 5x1m 28 9165 37 HiTrap Capto adhere 5x1m 28 4058 44 0 10 m 40 HiTrap IEX Selection Kit 7x1m 17 6002 33 HiTrap Desalting 5x5m 17 1408 01 0 05 _ 20 100 x 5 ml 11 0003 29 HiPrep 26 10 Desalting 1 53 ml 17 5087 01 o 4 o 4 53 ml 17 5087 02 T T T 10 20 30 mit Superloop 10 ml L 8 1113 83 Superloop 50 ml 1 18 1113 84 Troubleshooting Superloop 150 ml 1 18 1023 85 High backpressure Pack size available by special order e Column clogged Clean the column according to instructions Make sure the sample has been centrifuged and or filtered through a 0 45 um filter System clogged Replace the column with a piece of tubing Check pressure If backpressure gt 0 3 MPa clean system according to manual No binding Check tha
3. 1 0 M NaCl pH 7 0 Start buffer port A1 20 mM sodium citrate pH 3 0 Elution buffer port B 20 mM sodium citrate 1 0 M NaCl pH 3 0 2 Preparing the sample e diluting the sample in binding buffer or by buffer exchange using HiTrap Desalting or HiPrep 26 10 Desalting b Pass the sample through a 0 45 um filter 3 Preparing the system a Place the inlet tubing from port A1 8 port valve in the binding buffer and the tubing from port B 2 port valve in the elution buffer b Place the three brown waste tubings in waste c Connect the column between port 1 on the injection valve 7 port valve and the UV flow cell see Ordering information on next page for suitable columns d Fill the fraction collector rack with 18 mm tubes minimum 40 and position the white plate on the fractionation arm against the first tube e Connect a sample loop large enough for your sample between port 2 and 6 on the injection valve Use a syringe to manually fill the loop Note If a Superloop is needed additional information is supplied in the instructions for Superloop 11 0027 48 AC 2007 09 e p34 a Adjust the sample to composition of binding buffer by 4 Selecting Application Template and starting the method a Check the communication to PrimeView At the lower right corner of the screen the text Controlled By prime should be displayed b Use the arrow and OK buttons
4. 4 Selecting Application Template and starting the method a Check the communication to PrimeView At the lower right corner of the screen the text Controlled By prime should be displayed b Use the arrow and OK buttons to move in the menu tree until you find His Tag Purification HisTrap Set Sample Inj Vol Templates gt 00 0 mi 60 0 y Y Run Application Template Application Template T Press OK to start v M His Tag Purification HisTrap Run data displayed c Enter the sample volume and press OK to start the template Note If a 5 ml column is preferred see cue card on p 36 Theoretical gradient in His Tag Purification HisTrap Application Template B 100 Wash Priming Elution 50 4 Equilibration Sample Buffer wash Re equilibration 2 10 20 20 17 5 Min Total separation time 74 min sample application time 5 Typical result Ordering information Sample Clarified homogenate of E coli expressing Product Quantity Code No histidine tagged protein _rere sszZzz r Column HisTrap HP 1 ml HisTrap HP 5x1m 7 5247 01 Binding buffer port A1 20 mM phosphate 0 5 M NaCl 20 mM P A 4 imidazole pH 7 4 00x1m 7 5247 05 Elution buffer port B 20 mM phosphate 0 5 M NaCl 0 5 M ui 17 imidazole pH 7 4 isTrap FF 5x1m 7 5319 01 AUz80nm B 100 x 1 mI 17 5319 02 100 HisTrap FF crude 5x
5. B 26 100 20 80 15 160 1 04 40 05 20 of 0 7 1 r r r r r 0 5 wo 8615 20 235 30 min Troubleshooting High backpressure e Column clogged Clean the column according to instructions Make sure the sample has been centrifuged and or filtered through a 0 45 um filter System clogged Replace the column with a piece of tubing Check pressure If backpressure gt 0 3 MPa clean system according to manual No binding Regenerate the column with 3 column volumes CV water 3 CV 0 5 M NaOH 3 CV water 5 CV binding buffer before starting the run e Check that the correct column is used e Check that the inlet tubing from each buffer is connected to the correct inlet port e Check that the composition and pH of the buffers are correct Check that the sample has been adjusted to the binding buffer conditions e Check that your sample contains target protein No elution Check that the inlet tubing from each buffer is connected to the correct inlet port e Check that the composition and pH of the buffers are correct e Use alternative elution conditions according to the column instructions e Check that your sample contains target protein Ordering information Product Quantity Code No MBPTrap HP 5x1ml 28 9187 78 HiTrap Desalting 5x5ml 17 1408 01 100 x 5 ml 11 0003 29 HiPrep 26 10 Desalting 1 53 ml 17 5087 01 4 53 ml 17 5087 02 Superloop 10 ml 1 18 1113 83 Superloop 50 ml 1 18 1113
6. High backpressure Column clogged Clean the column according to instructions Make sure the sample has been centrifuged and or filtered through a 0 45 um filter e System clogged Replace the column with a piece of tubing Check pressure If backpressure gt 0 3 MPa clean system according to manual No binding e Check that the correct column is used Check that the inlet tubing from each buffer is connected to the correct inlet port e Check that the composition and pH of the buffers are correct e Check that the sample has been adjusted to binding buffer conditions e Check that your sample contains target protein No elution Check that the inlet tubing from each buffer is connected to the correct inlet port e Check that the composition and pH of the buffers are correct e Use alternative elution conditions according to the column instructions e Check that your sample contains target protein Ordering information Product Quantity Code No HiTrap IgM Purification HP 5x1ml 17 5110 01 HiTrap Desalting 5x5ml 17 1408 01 100 x 5 ml 11 0003 29 HiPrep 26 10 Desalting 1 53 ml 17 5087 01 4 53 ml 17 5087 02 Superloop 10 ml 1 18 1113 83 Superloop 50 ml IL 18 1113 84 Superloop 150 ml 1 18 1023 85 Pack size available by special order 11 0027 48 AC 2007 09 e p27 Albumin removal 1 Preparing the buffers Use high purity water and chemicals Filter all buffers through
7. Superloop 50 ml 1 18 1113 84 clean system according to manual Eluted sample still contaminated e Check that the sample load limit is not exceeded 11 0027 48 AC 2007 09 p7 Histidine tagged protein purification step elution 1 Preparing the buffers e Use high purity water and chemicals e Filter all buffers through a 0 45 um filter before use Binding buffer port A1 20 mM sodium phosphate 0 5 M NaCl 20 mM imidazole pH 7 4 Elution buffer port B 20 mM sodium phosphate 0 5 M NaCl 0 5 M imidazole pH 7 4 Prepare at least 500 ml of each eluent Alternative binding buffers 5 40 mM imidazole can be included in the binding buffer to reduce unspecific binding of non histidine tagged proteins The concentration of imidazole is protein dependent and if the protein of interest elutes or does not bind at a certain imidazole concentration then reduce the concentration 2 Preparing the sample a Adjust the sample to composition of binding buffer by diluting the sample in binding buffer or by buffer exchange using HiTrap Desalting or HiPrep 26 10 Desalting b Pass the sample through a 0 45 um filter Note If HisTrap FF crude column is used no filtration nor clarification of the sample is needed 3 Preparing the system a Place the inlet tubing from port A1 8 port valve in the binding buffer and the tubing from port B 2 port valve in the elution buffer b Place the three brown waste tubings in w
8. The Method Templates provides an easy and fast way to build up a method By setting defined parameters and volumes for each part of the chromatographic phase a method is built up Code No Column vol ml Affinity 17 0402 01 HiTrap Protein A HP 1 ml 17 0403 01 HiTrap Protein A HP 5 ml 17 5079 01 HiTrap rProtein A FF 1 ml 17 5080 01 HiTrap rProtein A FF 5 ml 17 0404 01 HiTrap Protein G HP 1 ml 17 0405 01 HiTrap Protein G HP 5 ml 11 0034 93 HiTrap MabSelect SuRe 1 ml 11 0034 94 HiTrap MabSelect SuRe 5 ml 28 4082 53 HiTrap MabSelect 1 ml 28 4082 55 HiTrap MabSelect 5 ml 28 4082 58 HiTrap MabSelect Xtra 1 ml 28 4082 60 HiTrap MabSelect Xtra 5 ml 28 9187 78 BPTrap 28 9187 79 BPTrap 17 5130 01 GSTrap FF 1 ml 17 5131 01 GSTrap FF 5 ml 28 4017 45 GSTrap 4B 1m 28 4017 47 GSTrap 4B 5 m 7 5110 01 HiTrap IgM Purification 1 ml 17 5111 01 HiTrap Ig Purification 5 ml 7 5247 01 HisTrap HP 1 ml 17 5248 02 HisTrap HP 5 ml 7 5319 01 HisTrap FF 1 m 17 5255 01 HisTrap FF 5 m 11 0004 58 HisTrap FF crude 1 ml 17 5286 01 HisTrap FF crude 5 ml 17 5281 01 GSTrap HP 1 m 17 5282 01 GSTrap HP 5 m HP 1 ml HP 5 ml 28 9075 46 StrepTrap HP 1 ml 28 9075 47 StrepTrap HP 5 ml 17 0920 03 HiTrap IM 17 0920 05 HiTrap IM 17 0921 02 HiTrap IM 17 0921 04 HiTrap IM AC HP 1 ml AC HP 5 ml AC FF 1 ml AC FF 5 ml 17 0408 01 HiTrap Chelating HP 1 ml 17 0409 03 HiTrap Ch
9. A HP 2x1m 17 0402 03 a i 280 nm 5x1m 17 0402 01 p pala 70 HiTrap rProtein A FF 2x1m 17 5079 02 5x1m 17 5079 01 ora HiTrap MabSelect SuRe 5x1m 11 0034 93 TE Mouse IgG L so HiTrap MabSelect 5x1m 28 4082 53 oS HiTrap MabSelect Xtra 5x1m 28 4082 58 40 HiTrap Desalting 5x5m 17 1408 01 sa 100 x 5 mI 11 0003 29 0 al t 3 l l i l T HiPrep 26 10 Desalting 1 53 ml 17 5087 01 10 20 30 40 50 min 4 53 ml 17 5087 02 Superloop 10 ml 1 18 1113 83 Troubleshooting Superloop 50 ml 1 18 1113 84 Superloop 150 ml 1 18 1023 85 High backpressure e Column clogged Clean the column according Pack size available by special order to instructions Make sure the sample has been centrifuged and or filtered through a 0 45 um filter e System clogged Replace the column with a piece of tubing Check pressure If backpressure gt 0 3 MPa clean system according to manual No binding e Check that the correct column is used e Check that the inlet tubing from each buffer is connected to the correct inlet port e Check that the composition and pH of the buffers are correct e Check that the sample has been adjusted to binding buffer conditions e Check that your sample contains target protein No elution e Check that the inlet tubing from each buffer is connected to the correct inlet port e Check that the composition and pH of the buffers are correct e Use alternative elution conditions according to the column instruct
10. FF 5 ml 7 0978 13 HiTrap Butyl S FF 1 ml 17 0978 14 HiTrap Butyl S FF 5 ml 28 4110 01 HiTrap Butyl HP 1 ml 28 4110 05 HiTrap Butyl HP 5 ml 17 5095 01 HiPrep 16 10 Phenyl FF high sub 7 5094 01 HiPrep 16 10 Phenyl FF low sub 17 5097 0 HiPrep 16 10 Octyl FF 7 5096 0 HiPrep 16 10 Butyl FF 17 1085 01 HiLoad 16 10 Phenyl Sepharose HP Gel filtration 7 1165 01 HiPrep 16 60 Sephacryl S 100 17 1194 01 HiPrep 26 60 Sephacryl S 100 7 1166 01 HiPrep 16 60 Sephacryl S 200 17 1195 01 HiPrep 26 60 Sephacryl S 200 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 Flow MPa wN NNNNA Frac vol ml min nananana wo ununun u Cal DUIUDALUIU DU DUuUDUDU Equil vol Wash1 vol ml 100 100 100 100 100 100 25 5 25 5 25 5 25 5 25 5 25 5 25 100 100 100 100 100 250 250 250 650 250 650 250 250 250 250 250 250 ml 40 40 40 40 40 40 N N N N o NOoONONOINONO gt BE E 6 sd Di Oo O D O oO oO e o oo OOOO Elu vol ml 400 400 400 400 400 400 20 00 20 00 20 00 20 00 20 00 20 00 20 00 400 400 400 400 400 80 480 80 480 80 480 80 480 80 480 80 480 Wash2 ml 100 100 100 100 100 100 25 25 25 25 25 2
11. contains target protein 11 0027 48 AC 2007 09 e p11 IMAC purification any metal 1 Preparing the buffers e Use high purity water and chemicals Filter all buffers through a 0 45 um filter before use Binding buffer port A1 20 mM sodium phosphate 0 5 M NaCl 20 mM imidazole pH 7 4 Wash eluent port A2 Distilled water Metal loading eluent port A3 100 mM metal salt solution metal chloride or metal sulphate e g 100 mM CuSO 100 MM CoCl or 100 mM ZnCl in distilled H O Elution buffer port B 20 mM sodium phosphate 0 5 M NaCl 0 5 M imidazole pH 7 4 Prepare at least 500 ml of each eluent Alternative buffers 5 40 mM imidazole can be included in the binding buffer to reduce unspecific binding of non histidine tagged proteins The concentration of imidazole is protein dependent and if the protein of interest elutes or does not bind at a certain imidazole concentration then reduce the concentration 2 Preparing the sample e diluting the sample in binding buffer or e by buffer exchange using HiTrap Desalting or HiPrep 26 10 Desalting b Pass the sample through a 0 45 um filter 3 Preparing the system a Place each inlet tubing from port A 8 port valve in eluents as given above and the tubing from port B 2 port valve in the elution buffer b Place the three brown waste tubings in waste c Connect the column between port 1 on the injection valve 7 port valve and the U
12. e Column clogged Clean the column according to instructions Make sure the sample has been centrifuged and or filtered through a 0 45 um filter System clogged Replace the column with a piece of tubing Check pressure If backpressure gt 0 3 MPa clean system according to manual No binding Check that the correct column is used e Check that the inlet tubing from each buffer is connected to the correct inlet port e Check that the composition and pH of the buffers are correct e Check that the sample has been adjusted to binding buffer conditions e Check that your sample contains target protein No elution e Check that the inlet tubing from each buffer is connected to the correct inlet port e Check that the composition and pH of the buffers are correct e Use alternative elution conditions according to the column instructions e Check that your sample contains target protein Ordering information Product Quantity HisTrap HP 5x1ml 100 x 1 mI HisTrap FF 5x1ml 100 x 1 mI HiTrap Desalting 5x5ml 100 x 5 ml HiPrep 26 10 Desalting 1 53 ml 4 53 ml Superloop 10 ml 1 Superloop 50 ml Superloop 150 ml 1 Pack size available by special order Code No 17 5247 0 17 5247 05 17 5319 0 17 5319 02 17 1408 01 11 0003 29 17 5087 0 17 5087 02 18 1113 83 18 1113 84 18 1023 85 11 0027 48 AC 2007 09 e p15 GST tagged protein purification 1 Preparing the buffers e Use
13. to move in the menu tree until you find Cation Exchange HiTrap SP Set Sample Inj Vol Templates 00 0 m 00 0 A4 Y Application Template Run Application Template T Press OK to start M Cation Exchange A HiTrap SP g Run data displayed c Enter the sample volume and press OK to start the template Note If a 5 ml column is preferred see cue card on p 36 Theoretical gradient in Cation Exchange HiTrap SP Application Template B Wash 2 100 7 Priming amp Equilibration Elution 50 Sample Re equilibration gt 11 10 20 17 5 Min Total separation time 63 min sample application time 5 Typical result Ordering information Sample Protein mix containing chymotrypsinogen A cytochrome C Product Quantity Code No and lysozyme in start buffer i A giSFNA amp iilxgx L E iii Column HiTrap SP HP 1 ml HiTrap SP HP 5x1m 17 1151 01 Start buffer A1 20 mM sodium acetate pH 5 5 n Elution buffer B 20 mM sodium acetate 1 0 M NaCl pH 5 5 HiTrap SP FF 5x1m 17 5054 01 AUze0nm a HiTrap CM FF 5x1m 17 5056 01 sl Programmed 48 HiTrap SP XL 5x1m 17 5160 01 f HiTrap Capto S 5x1m 17 5441 22 03 HiTrap Capto MMC 5x1m 11 0032 73 HiTrap IEX Selection Kit Txim 17 6002 33 0 2 HiTrap Desalting 5x5m 17 1408 01 100 x 5 ml 11 0003 29 01 4 HiPrep 26 10 Desalting 1 53 ml 17 5087 01 4 53 ml 17 5087 02 0 Superloop 10 ml 1 18 1113 83 Superloop 50 ml 1 18 1113 84 0 10 15 20 25 30 min Superlo
14. 113 83 la 0 Superloop 50 ml 1 18 1113 84 T T T 4 4 d 125 25 min Superloop 150 ml 8 1023 85 Troubles hooting Pack size available by special order High backpressure e Column clogged Clean the column according to instructions Make sure the sample has been centrifuged and or filtered through a 0 45 um filter System clogged Replace the column with a piece of tubing Check pressure If backpressure gt 0 3 MPa clean system according to manual No binding e Check that the correct column is used e Check that the inlet tubing from each buffer is connected to the correct inlet port e Check that the composition and pH of the buffers are correct e Check that the sample has been adjusted to binding buffer conditions e Check that your sample contains target protein No elution e Check that the inlet tubing from each buffer is connected to the correct inlet port e Check that the composition and pH of the buffers are correct e Use alternative elution conditions according to the column instructions e Check that your sample contains target protein 11 0027 48 AC 2007 09 e p17 Strep l tagged protein purification 1 Preparing the buffers e Use high purity water and chemicals Filter all buffers through a 0 45 um filter before use Binding buffer port A1 100 mM Tris HCl 150 mM NaCl 1 mM EDTA pH 8 0 Elution buffer port B 100 mM Tris HCl 150 mM NaCl 1 mM EDTA 2 5 mM desthiobiotin p
15. 1m 11 0004 58 UV 280 nm Programmed B 100x1ml 11 0004 59 4 M 80 HiTrap Desalting 5x5m 17 1408 01 L 60 100 x 5 ml 11 0003 29 104 HiPrep 26 10 Desalting 1 53 ml 17 5087 01 FS 4 53 ml 17 5087 02 0 54 A Superloop 10 ml 1 18 1113 83 Superloop 50 ml 1 18 1113 84 O Superloop 150 ml 1 18 1023 85 0 10 20 30 40 50 60 min TTT Pack size available by special order Troubleshooting High backpressure e Column clogged Clean the column according to instructions Make sure the sample has been centrifuged and or filtered through a 0 45 um filter For unclarified samples using HisTrap FF crude make sure the sample has been lysed properly e g using thorough sonication and DNase treatment System clogged Replace the column with a piece of tubing Check pressure If backpressure gt 0 3 MPa clean system according to manual No binding Check that the correct column is used e Check that the inlet tubing from each buffer is connected to the correct inlet port e Check that the composition and pH of the buffers are correct e Check that the sample has been adjusted to binding buffer conditions e Check that your sample contains target protein No elution e Check that the inlet tubing from each buffer is connected to the correct inlet port e Check that the composition and pH of the buffers are correct e Use alternative elution conditions according to the column instructions Check that your sample
16. 5 25 100 100 100 100 100 The pressure limits 0 35 and 0 5 MPa respectively includes the pressure limit of the column 0 15 and 0 3 MPa respectively and the 7 1167 01 HiPrep 16 60 Sephacryl S 300 17 1196 01 HiPrep 26 60 Sephacryl S 300 7 1139 01 HiLoad 16 60 Superdex 30 pg 17 1140 01 HiLoad 26 60 Superdex 30 pg 7 1068 01 HiLoad 16 60 Superdex 75 pg 17 1070 01 HiLoad 26 60 Superdex 75 pg 7 1069 01 HiLoad 16 60 Superdex 200 pg 17 1071 01 HiLoad 26 60 Superdex 200 pg backpressure that the flow restrictor after the column creates 0 2 MPa Note Don t forget to check that you have a sufficient number of tubes for fractionation The templates are pre set to fractionate during the elution and wash2 phases 11 0027 48 AC 2007 09 e p38 11 0027 48 AC 2007 09 e p39 GE imagination at work and GE monogram are trade marks of General Electric Company Capto MabSelect MabSelect Xtra MabSelect SuRe Drop Design GSTPrep GSTrap HisPrep HisTrap HiPrep HiLoad HiTrap MBPTrap StrepTrap PrimeView Sephacryl Super dex Superloop AKTA and AKTAprime are trademarks o GE Healthcare companies Capto Q Separating viral particles with Capto Q products may require a license under United States patent number 6 537 793 B2 and equivalent patents and patent applica tions in other countries owned by Centelion SAS Such a license is not included with the purchase of Capto Q but is included with the purchase of Capto
17. 7 03 HiTrap Heparin HP 5 ml 0 5 5 2 5 25 50 25 0 17 5112 01 HiTrap Streptavidin HP 1 ml 0 5 1 0 5 10 10 10 0 7 5143 01 HiTrap Benzamidine FF high sub 1 ml 0 5 1 0 5 5 20 5 0 7 5144 01 HiTrap Benzamidine FF high sub 5 ml 0 5 5 25 25 100 25 0 7 5189 0 HiPrep 16 10 Heparin FF 0 5 5 5 00 200 100 0 17 0921 06 HiPrep IMAC FF 16 10 0 5 5 5 100 200 100 0 7 5234 01 GSTPrep FF 16 10 0 5 1 5 5 00 400 100 0 17 5256 01 HisPrep M FF 16 10 0 5 15 5 100 400 100 0 Buffer exchange 17 1408 01 HiTrap Desalting 5 ml 0 5 5 0 5 15 0 7 HiTrap Desalting 2 x 5 ml 0 5 5 1 30 0 14 0 7 5087 0 HiPrep 26 10 Desalting 0 35 10 2 5 00 0 80 lon exchange 7 6002 33 HiTrap IEX Selection Kit 1 ml 0 5 5 2 20 5 17 1153 0 HiTrap Q HP 1 m 0 5 1 5 2 20 5 7 1154 0 HiTrap Q HP 5 m 0 5 5 5 25 10 00 25 17 5053 0 HiTrap Q FF 1 ml 0 5 1 5 2 20 5 7 5156 0 HiTrap Q FF 5 ml 0 5 5 5 25 10 00 25 17 5055 0 HiTrap DEAE FF 1 ml 0 5 1 1 5 2 20 5 7 5154 0 HiTrap DEAE FF 5 ml 0 5 5 5 25 10 00 25 17 5162 0 HiTrap ANX FF high sub 1 ml 0 5 1 5 2 20 5 7 5163 0 HiTrap ANX FF high sub 5 ml 0 5 5 5 25 10 00 25 17 5158 0 HiTrap Q XL 1m 0 5 1 1 5 2 20 5 7 5159 0 HiTrap Q XL 5m 0 5 5 5 25 10 00 25 17 1151 0 HiTrap SP HP 1 ml 0 5 1 5 2 20 5 7 1152 0 HiTrap SP HP 5 ml 0 5 5 5 25 10 00 25 17 5054 01 HiTrap SP FF 1 ml 0 5 1 1 5 2 20 5 7 5157 0 HiTrap SP FF 5 ml 0 5 5 5 25 10 00 25 7 5056 0 HiTrap CM FF 1 ml 0 5 5 2 20 5 17 5155 01 HiTrap CM FF 5 ml 0 5 5 5 25 10 100 25 7 5160 0 HiTr
18. 84 Superloop 150 ml i 18 1023 85 Pack size available by special order 11 0027 48 AC 2007 09 e p21 MAD purification step elution 1 Preparing the buffers Use high purity water and chemicals Filter all buffers thtough a 0 45 um filter before use Binding buffer port A1 20 mM sodium phosphate pH 7 0 Elution buffer port B 0 1 M glycine HCl pH 3 0 Prepare at least 500 ml of each eluent With some antibodies e g mouse IgG it might be necessary to add NaCl up to 3 M in the binding buffer to achieve efficient binding when using HiTrap Protein A HP and HiTrap rProtein A FF 2 Preparing the sample a e Adjust the sample to composition of binding buffer by diluting the sample with binding buffer or by buffer exchange using HiTrap Desalting or HiPrep 26 10 Desalting Pass the sample through a 0 45 um filter Preparing the system Place the inlet tubing from port A1 8 port valve in the binding buffer and the tubing from port B 2 port valve in the elution buffer Place the three brown waste tubings in waste Connect the column between port 1 on the injection valve 7 port valve and the UV flow cell see Ordering information on next page for suitable columns Fill the fraction collector rack with 18 mm tubes minimum 10 and position the white plate on the fractionation arm against the first tube Connect a sample loop large enough for your sample between port 2 and 6 on the injection
19. Binding buffer port A1 20 mM Tris HCI 200 mM NaCl 1 mM EDTA 1 mM DTT pH 7 4 Elution buffer port B 20 mM Tris HCI 200 mM NaCl 1 mM EDTA 1 mM DTT 10 mM maltose pH 7 4 Prepare at least 500 ml of each eluent 2 Preparing the sample a Adjust the sample to composition of binding buffer by e diluting the sample in binding buffer or by buffer exchange using HiTrap Desalting or HiPrep 26 10 Desalting b Pass the sample through a 0 45 um filter 3 Preparing the system a Place the inlet tubing from port A1 8 port valve in the binding buffer and the tubing from port B 2 port valve in the elution buffer b Place the three brown waste tubings in waste c Connect the column between port 1 on the injection valve 7 port valve and the UV flow cell see Ordering information on next page for suitable columns d Fill the fraction collector rack with 18 mm tubes minimum 10 and position the white plate on the fractionation arm against the first tube e Connect a sample loop large enough for your sample between port 2 and 6 on the injection valve Use a syringe to manually fill the loop Note If a Superloop is needed additional information is supplied in the instructions for Superloop The number of tubes to insert in the fraction collector varies with the sample volume Fill the fraction collector with 20 tubes one tube ml sample For example if the sample volume is 10 ml fill the fractio
20. GE Healthcare AKTAprime plus Cue Cards Contents System Preparation Buffer exchange on HiTrap Desalting Buffer exchange on HiPrep 26 10 Desalting Histidine tagged protein purification step elution Histidine tagged protein purification gradient elution IMAC purification any metal On column refolding GST tagged protein purification Strep Il tagged protein purification MBP tagged protein purification MAb purification step elution MAb purification gradient elution IgM purification Albumin removal Removal of trypsin like serine proteases Anion exchange Cation exchange Method Templates value table 10 12 14 16 18 20 22 24 26 28 30 32 34 36 KTA System Preparation 1 Starting the system Press the button on the back of the system a b Wait until the system self test has been completed 30 40 seconds Templates is displayed when the system is ready to use 2 Starting the computer Start the computer a b c Check the communication between system and computer Start PrimeView Tip If communication has been established the text Controlled By prime should be displayed at the lower right corner of the screen If not check the connections 3 Purging the system flow path If the flow path is filled with storage solution e g ethanol 20 remove it as follows a e Place the three brown waste tubings in waste e Set all inlet tubings B A1 that will
21. H 8 0 Prepare at least 500 ml of each eluent 2 a e Preparing the sample Adjust the sample to composition of binding buffer by diluting the sample in binding buffer or by buffer exchange using HiTrap Desalting or HiPrep 26 10 Desalting Pass the sample through a 0 45 um filter Preparing the system Place the inlet tubing from port A1 8 port valve in the binding buffer and the tubing from port B 2 port valve in the elution buffer Place the three brown waste tubings in waste Connect the column between port 1 on the injection valve 7 port valve and the UV flow cell see Ordering information on next page for suitable columns Fill the fraction collector rack with 18 mm tubes minimum 10 and position the white plate on the fractionation arm against the first tube Connect a sample loop large enough for your sample between port 2 and 6 on the injection valve Use a syringe to manually fill the loop Note If a Superloop is needed additional information is supplied in the instructions for Superloop The number of tubes to insert in the fraction collector varies with the sample volume Fill the fraction collector with 20 tubes one tube ml sample For example if the sample volume is 10 ml fill the fraction collector with 20 10 30 tubes However note that the maximum capacity of the fraction collector is 95 tubes limiting the sample volume to 75 ml 11 0027 48 AC 2007 09 e p18 4 Selecting Appli
22. Preparing the buffers e Use high purity water and chemicals e Filter all buffers through a 0 45 um filter before use Binding buffer port A1 100 mM sodium phosphate 100 mM sodium citrate pH 7 0 Elution buffer port B 100 mM sodium phosphate 100 mM sodium citrate pH 3 0 Prepare at least 500 ml of each eluent With some antibodies e g mouse IgG it might be necessary to add NaCl up to 3 M in the binding buffer to achieve efficient binding when using HiTrap Protein A HP and HiTrap rProtein A FF 2 Preparing the sample a Adjust the sample to composition of binding buffer by e diluting the sample in binding buffer or e by buffer exchange using HiTrap Desalting or HiPrep 26 10 Desalting b Pass the sample through a 0 45 um filter 3 Preparing the system a Place the inlet tubing from port A1 8 port valve in the binding buffer and the tubing from port B 2 port valve in the elution buffer b Place the three brown waste tubings in waste c Connect the column between port 1 on the injection valve 7 port valve and the UV flow cell see Ordering information on next page for suitable columns d Fill the fraction collector rack with 18 mm tubes minimum 40 and position the white plate on the fractionation arm against the first tube e Connect a sample loop large enough for your sample between port 2 and 6 on the injection valve Use a syringe to manually fill the loop Note If a Superloo
23. System clogged Replace the column with a piece of tubing Check pressure If backpressure gt 0 3 MPa clean system according to manual No binding e Regenerate the column with 3 column volumes CV water 3 CV 0 5 M NaOH 3 CV water 5 CV binding buffer before starting the run Check that the correct column is used e Check that the inlet tubing from each buffer is connected to the correct inlet port e Check that the composition and pH of the buffers are correct e Check that the sample has been adjusted to the binding buffer conditions e Check that your sample contains target protein No elution e Check that the inlet tubing from each buffer is connected to the correct inlet port e Check that the composition and pH of the buffers are correct e Use alternative elution conditions according to the column instructions e Check that your sample contains target protein Ordering information Product Quantity StrepTrap HP 5x1ml HiTrap Desalting 5x5ml 100 x 5 ml HiPrep 26 10 Desalting 1 53 ml 4 53 ml Superloop 10 ml i Superloop 50 ml 1 Superloop 150 ml 1 Pack size available by special order Code No 28 9075 46 17 1408 01 11 0003 29 17 5087 01 17 5087 02 18 1113 83 18 1113 84 18 1023 85 11 0027 48 AC 2007 09 e p19 MBP tagged protein purification 1 Preparing the buffers e Use high purity water and chemicals Filter all buffers through a 0 45 um filter before use
24. V flow cell see Ordering information on next page for suitable columns d Fill the fraction collector rack with 18 mm tubes minimum 40 and position the white plate on the fractionation arm against the first tube e Connect a sample loop large enough for your sample between port 2 and 6 on the injection valve Use a syringe to manually fill the loop Note If a Superloop is needed additional information is supplied in the instructions for Superloop 11 0027 48 AC 2007 09 e p12 a Adjustthe sample to composition of binding buffer by 4 Selecting Application Template and a b starting the method Check the communication to PrimeView At the lower right corner of the screen the text Controlled By prime should be displayed Use the arrow and OK buttons to move in the menu tree until you find IMAC Purification Uncharged HiTrap Set Sample Inj Vol Templates gt 00 0 ml 00 0 Y Application Template Run Application Template T Press OK to start Y Y IMAC Purification Uncharged HiTrap Run data displayed c Enter the sample volume and press OK to start the template Note If a 5 ml column is preferred see cue card on p 36 Theoretical gradient in IMAC Purification Uncharged HiTrap Application Template 100 B Wash Priming amp water wash Metal loading Elution Priming amp water wash Equilibration Buffer wash Sam
25. ViralQ products Capto ViralQ With the purchase of Capto ViralQ the customer is granted a free limited license under US patent 6 537 793 B2 and equivalent patents and patent applications in other countries owned by Centelion SAS to separate viral particles solely through use of the product purchased StrepTrap HP This product has been manufactured by GE Healthcare and contains Strep Tactin manufactured by BA GmbH which has been immobilized to GE Health care s chromatography media Strep Tactin is covered by US patent number 6 103 493 and equivalent patents and patent applications in other countries The purchase of StrepTrap HP includes a license under such patents imited to internal use but not re sale Please contact IBA or further information on licenses for commercial use of Strep Tactin Histidine tagged protein purification Purification and preparation of fusion proteins and affinity peptides comprising at least two adjacent histidine residues may require a license under US patent numbers 5 284 933 and 5 310 663 and equivalent patents and patent applications in other countries assignee Hoffman La Roche Inc For contact information for your local office please visit www gelifesciences com contact GE Healthcare Bio Sciences AB Bj rkgatan 30 SE 751 84 Uppsala Sweden www gelifesciences com protein purification imagination at work 2007 General Electric Company All rights reserved Fir
26. a 0 45 um filter before use Binding buffer port A1 20 mM sodium phosphate pH 7 0 Elution buffer port B 20 mM sodium phosphate 2 0 M NaCl pH 7 0 Prepare at least 500 ml of each eluent 2 a e Preparing the sample Adjust the sample to composition of binding buffer by diluting the sample in binding buffer or by buffer exchange using HiTrap Desalting or HiPrep 26 10 Desalting Pass the sample through a 0 45 um filter Preparing the system Place the inlet tubing from port A1 8 port valve in the binding buffer and the tubing from port B 2 port valve in the elution buffer Place the three brown waste tubings in waste Connect the column between port 1 on the injection valve 7 port valve and the UV flow cell see Ordering information on next page for suitable columns Fill the fraction collector rack with 18 mm tubes minimum 20 and position the white plate on the fractionation arm against the first tube Connect a sample loop large enough for your sample between port 2 and 6 on the injection valve Use a syringe to manually fill the loop Note If a Superloop is needed additional information is supplied in the instructions for Superloop 11 0027 48 AC 2007 09 p28 4 a b Selecting Application Template and starting the method Check the communication to PrimeView At the lower right corner of the screen the text Controlled By prime should be displayed Use the arrow and OK butt
27. ap SP XL 1 ml 0 5 5 2 20 5 17 5161 01 HiTrap SP XL 5 ml 0 5 5 5 25 10 100 25 1 0013 02 HiTrap Capto Q 1 ml 0 5 5 2 20 5 11 0013 03 HiTrap Capto Q 5 ml 0 5 5 5 25 10 100 25 28 9078 09 HiTrap Capto ViralQ 5 ml 0 5 5 5 25 0 00 25 11 0032 73 HiTrap Capto MMC 1 ml 0 5 1 1 5 2 20 5 1 0032 75 HiTrap Capto MMC 5 ml 0 5 5 5 25 0 100 25 17 5441 22 HiTrap Capto S 1 ml 0 5 1 1 5 2 20 5 7 5441 23 HiTrap Capto S 5 ml 0 5 5 5 25 0 100 25 28 4058 44 HiTrap Capto adhere 1 ml 0 5 1 i 5 2 20 5 28 4058 46 HiTrap Capto adhere 5 ml 0 5 5 5 25 0 100 25 28 9165 37 HiTrap Capto DEAE 1 ml 0 5 1 1 5 2 20 5 28 9165 40 HiTrap Capto DEAE 5 ml 0 5 5 5 25 0 100 25 17 5190 01 HiPrep 16 10 Q FF 0 5 5 5 100 40 400 100 17 5192 01 HiPrep 16 10 SP FF 0 5 5 5 100 40 400 100 The table continues on next page 11 0027 48 AC 2007 09 e p37 Code No Column Pressure vol ml 7 5092 01 HiPrep 16 10 Q XL 17 5093 01 HiPrep 16 10 SP XL 7 5091 0 HiPrep 16 10 CM FF 17 5090 01 HiPrep 16 10 DEAE FF 7 1064 01 HiLoad 16 10 Q Sepharose HP 17 1137 01 HiLoad 16 10 SP Sepharose HP Hydrophobic interaction 17 1355 01 HiTrap Phenyl FF high sub 1ml 7 5193 01 HiTrap Phenyl FF high sub 5ml 7 1353 01 HiTrap Phenyl FF low sub 1ml 7 5194 01 HiTrap Phenyl FF low sub 5ml 7 1351 01 HiTrap Phenyl HP 1 ml 17 5195 01 HiTrap Phenyl HP 5 ml 7 1359 01 HiTrap Octyl FF 1 ml 17 5196 01 HiTrap Octyl FF 5 ml 7 1357 01 HiTrap Butyl FF 1 ml 17 5197 01 HiTrap Butyl
28. aste c Connect the column between port 1 on the injection valve 7 port valve and the UV flow cell see Ordering information on next page for suitable columns d Fill the fraction collector rack with 18 mm tubes and position the white plate on the fractionation arm against the first tube The number of tubes to insert in the fraction collector varies with the sample volume Fill the fraction collector with 20 tubes one tube ml sample For example if the sample volume is 10 ml fill the fraction collector with 20 10 30 tubes However note that the maximum capacity of the fraction collector is 95 tubes limiting the sample volume to 75 ml 11 0027 48 AC 2007 09 e p8 e a b Connect a sample loop large enough for your sample between port 2 and 6 on the injection valve Use a syringe to manually fill the loop Note If a Superloop is needed additional information is supplied in the instructions for Superloop Selecting Application Template and starting the method Check the communication to PrimeView At the lower right corner of the screen the text Controlled By prime should be displayed Use the arrow and OK buttons to move in the menu tree until you find Affinity Purification any HiTrap Set Sample Inj Vol Templates gt 00 0 mh 60 0 y Y Application Template Run Application Template T Press OK to start v y Affinity Purification any HiTrap Run da
29. be used in water see the appropriate cue card e Select the application template System Wash Method select the inlets to be washed Press OK a b c 6 a Note Wait at least 15 minutes before using the system after the lamp has been turned on to avoid drifting UV base line If using pH Calibrate the pH The pH must be calibrated every day See AKTAprime plus User Manual for instructions Checking the flow rate Set the injection valve to position WASTE Start the flow 1 50 ml min Use a graduated glass cylinder to collect at the WASTE outlet port 5 Collect during at least one minute Note Inaccurate flow rate may be due to air in the pump If this is the case flush the pump with buffer and try again If the problem persists flush the system with ehtanol or methanol followed by water Preparing the fraction collector Check that the delay volume is correct The delay volume is equal to the volume between the UV flow cell and the fraction collector default 380 pil Set Delay UV to Frac Set Parameters Oc pl Select Buffer V Pos B a AERAN Templates Y Y Application Template A System Wash Method b If there are large amounts of air in the tubing Fill the tubing using Purge kit P 950 See KTAprime plus User Manual for detailed instructions 4 Preparing the monitors a Check the UV lamp filter position and the lamp pos
30. buffer exchange using HiTrap Desalting or HiPrep 26 10 Desalting b Pass the sample through a 0 45 um filter 3 Preparing the system a Place the inlet tubing from port A1 8 port valve in the binding buffer and the tubing from port B 2 port valve in the elution buffer b Place the three brown waste tubings in waste c Connect the column between port 1 on the injection valve 7 port valve and the UV flow cell see Ordering information on next page for suitable columns d Fill the fraction collector rack with 18 mm tubes minimum 40 and position the white plate on the fractionation arm against the first tube e Connect a sample loop large enough for your sample between port 2 and 6 on the injection valve Use a syringe to manually fill the loop Note If a Superloop is needed additional information is supplied in the instructions for Superloop 4 Selecting Application Template and starting the method Check the communication to PrimeView At the lower right corner of the screen the text Controlled By a 11 0027 48 AC 2007 09 e p30 prime should be displayed b Use the arrow and OK buttons to move in the menu tree until you find Affinity Purification any HiTrap c Enter the sample volume and press OK to start the template Note If a 5 ml column is preferred see cue card on p 36 Set Sample Inj Vol Templates 00 0 ml 00 0 ad P Run Application Template Application Te
31. cation Template and starting the method a Check the communication to PrimeView At the lower right corner of the screen the text Controlled By prime should be displayed b Use the arrow and OK buttons to move in the menu tree until you find Affinity Purification any HiTrap Teo O Y Application Template Run Application Template 7 Press OK to start Y M Affinity Purification 6 any Aap Run data displayed c Enter the sample volume and press OK to start the template Note If a 5 ml column is preferred see cue card on p 36 Theoretical gradient in Affinity Purification any HiTrap Application Template B 100 Elution Priming Equilibration Water wash amp 50 priming Sample Re equili 1 10 20 10 5 Min Total separation time 47 min sample application time 5 Typical result Sample Clarified lysate of E coli expressing StreplIl tagged protein Column StrepTrap HP 1 ml Binding buffer port A1 100 mM Tris HCI 150 mM NaCl 1 mM EDTA pH 8 0 Elution buffer port B 100 mM Tris HCI 150 mM NaCl 1 mM EDTA 2 5 mM desthiobiotin pH 8 0 AUza0 nm B a 100 20 80 15 60 104 Lao 0 5 20 0 N 0 0 5 10 15 20 25 30 35 min Troubleshooting High backpressure e Column clogged Clean the column according to instructions Make sure the sample has been centrifuged and or filtered through a 0 45 um filter
32. e gt 0 3 MPa clean system according to manual Pack size available by special order Eluted sample still contaminated e Check that the sample load limit is not exceeded 11 0027 48 AC 2007 09 e p5 Buffer exchange on HiPrep 26 10 Desalting 1 Preparing the buffers e Use high purity water and chemicals Filter all buffers through a 0 45 um filter before use Buffer port A1 20 mM sodium phosphate 0 15 M NaCl pH 7 0 Prepare at least 500 ml eluent When performing buffer exchange use the appropriate buffer 2 Preparing the sample Pass the sample through a 0 45 um filter The maximum recommended sample volume is 15 ml 3 Preparing the system a Place the inlet tubing from port A1 8 port valve and port B 2 port valve in the buffer b Place the three brown waste tubings in waste c Connect the column between port 1 on the injection valve 7 port valve and the UV flow cell see Ordering information on next page for suitable columns d Fill the fraction collector rack with 18 mm tubes minimum 25 and position the white plate on the fractionation arm against the first tube e Connect a sample loop large enough for your sample between port 2 and 6 on the injection valve Use a syringe to manually fill the loop Note If a Superloop is needed additional information is supplied in the instructions for Superloop 4 Selecting Application Template and starting the method a Check th
33. e communication to PrimeView At the lower right corner of the screen the text Controlled By prime should be displayed b Use the arrow and OK buttons to move in the menu tree until you find Desalting HiPrep Desalting Set Sample Inj Vol TRAPASS gt oom 60 0 y Y Application Template Run Application Template Press OK to start Y Y Desalting HiPrep Desalting Run data displayed 11 0027 48 AC 2007 09 e p6 c Enter the sample volume and press OK to start the template Theoretical gradient in Desalting HiPrep Desalting Application Template B 100 Priming amp Equilibration 50 Elution i Sample 13 5 Min Total separation time 18 min sample application time 5 Typical result Sample BSA and sodium chloride Column HiPrep 26 10 Desalting 53 ml Buffer A1 20 mM sodium phosphate 0 15 M NaCl pH 7 0 AUz80nm 05 7 BSA 04 74 x 034 0 2 UV 280 nm 0 1 Conductivity Troubleshooting Ordering information High backpressure Product Quantity Code No e Column clogged Clean the column according HiPrep 26 10 Desalting 1 53 ml 17 5087 01 to instructions Make sure the sample has been i 4 53 ml 17 5087 02 centrifuged and or filtered through a 0 45 um filter e System clogged Replace the column with a piece Superloop 10 ml 1 18 1113 83 of tubing Check pressure If backpressure gt 0 3 MPa
34. ected to the correct inlet port e Check that the composition and pH of the buffers are correct Check that the sample has been adjusted to binding buffer conditions e Check that your sample contains target protein No elution Check that the inlet tubing from each buffer is connected to the correct inlet port e Check that the composition and pH of the buffers are correct e Use alternative elution conditions according to the column instructions e Check that your sample contains target protein 11 0027 48 AC 2007 09 e p13 On column refolding 1 Preparing the buffers e Use high purity water and chemicals Filter all buffers through a 0 45 um filter before use Binding buffer port A1 6 M guanidine hydrochloride 20 mM Tris HCl 0 5 M NaCl 5 mM imidazole 1 mM 2 mercaptoethanol pH 8 0 Solubilisation buffer port A2 6 M urea 20 mM Tris HCl 0 5 M NaCl 5 mM imidazole 1 mM 2 mercaptoethanol pH 8 0 Elution buffer port A3 20 mM Tris HCl 0 5 M NaCl 0 5 M imidazole 1 mM 2 mercaptoethanol pH 8 0 Refolding buffer port B 20 mM Tris HCl 0 5 M NaCl 5 mM imidazole 1 mM 2 mercaptoethanol pH 8 0 Prepare at least 500 ml of each eluent Alternative binding buffers 5 40 mM imidazole can be included in the binding buffer to reduce unspecific binding of non histidine tagged proteins The concentration of imidazole is protein dependent and if the protein of interest elut
35. elating HP 5 ml 17 0412 01 HiTrap Blue HP 1 ml 7 0413 01 HiTrap Blue HP 5 ml The table continues on next page 1 0027 48 AC 2007 09 e p36 Pressure MPa 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 This table contains suggested values for a selection of columns which enables a fast start The values can then Flow ml min O on Pont A nu u i uw Frac vol ml 0 5 2 5 0 5 2 5 0 5 2 5 0 5 2 5 0 5 2 5 0 5 2 5 0 5 25 0 5 2 5 0 5 2 5 0 5 2 5 0 5 2 5 0 5 2 5 0 5 2 5 0 5 2 5 0 5 2 5 0 5 2 5 0 5 2 5 0 5 2 5 0 5 2 5 Equil vol Wash1 vol ml ml 10 50 10 50 10 50 10 50 10 50 10 50 15 50 20 100 20 100 20 100 20 100 20 100 20 100 10 50 10 50 20 100 20 100 20 100 10 50 be optimized to suit the specific application Elu vol ml 25 25 25 25 25 25 15 50 25 25 25 25 25 25 25 25 25 25 25 25 Wash2 ml oo Oo oo oOo Oo fo ooo oOo a fe oo oo coo Aa oe oa P _ aca oOo ace aco ao no coo Cc oOo So amp Code No Column Pressure Flow Frac vol Equilvol Wash1 vol Elu vol Wash2 vol ml MPa ml min ml ml ml ml ml 17 0406 01 HiTrap Heparin HP 1 ml 0 5 1 0 5 5 10 5 0 17 040
36. es or does not bind at a certain imidazole concentration then reduce the concentration Include the same imidazole concentration as in used binding buffer 2 Preparing the sample a Adjust the sample to composition of binding buffer by e diluting the sample in binding buffer or e by buffer exchange using HiTrap Desalting or HiPrep 26 10 Desalting b Pass the sample through a 0 45 um filter 3 Preparing the system a Place each inlet tubing from port A 8 port valve in eluents as given above and the tubing from port B 2 port valve in the elution buffer b Place the three brown waste tubings in waste c Connect the column between port 1 on the injection valve 7 port valve and the UV flow cell see Ordering information on next page for suitable columns d Fill the fraction collector rack with 18 mm tubes minimum 40 and position the white plate on the fractionation arm against the first tube e Connect a sample loop large enough for your sample between port 2 and 6 on the injection valve Use a syringe to manually fill the loop 11 0027 48 AC 2007 09 e p14 Note If a Superloop is needed additional information is supplied in the instructions for Superloop 4 Selecting Application Template and starting the method a Check the communication to PrimeView At the lower right corner of the screen the text Controlled By prime should be displayed b Use the arrow and OK buttons to move in the menu
37. ffer and the tubing from port B 2 port valve in the elution buffer b Place the three brown waste tubings in waste c Connect the column between port 1 on the injection valve 7 port valve and the UV flow cell see Ordering information on next page for suitable columns d Fill the fraction collector rack with 18 mm tubes minimum 40 and position the white plate on the fractionation arm against the first tube e Connect a sample loop large enough for your sample between port 2 and 6 on the injection valve Use a syringe to manually fill the loop Note If a Superloop is needed additional information is supplied in the instructions for Superloop 11 0027 48 AC 2007 09 e p32 4 Selecting Application Template and a b starting the method Check the communication to PrimeView At the lower right corner of the screen the text Controlled By prime should be displayed Use the arrow and OK buttons to move in the menu tree until you find Anion Exchange HiTrap Q Set Sample Inj Vol Templates gt 0 0m 60 0 y Y Application Template Run Application Template Press OK to start Y Y e Run data displayed c Enter the sample volume and press OK to start the template Note If a 5 ml column is preferred see cue card on p 36 Theoretical gradient in Anion Exchange HiTrap Q Application Template 100 T B Wash 2 Priming amp Equilibration Elution
38. glycine HCl pH 3 0 Hil rap rProtein A FF 2x1m 17 5079 02 5x1m 17 5079 01 HiTrap MabSelect SuRe 5x1m 11 0034 93 HiTrap MabSelect 5x1m 28 4082 53 HiTrap MabSelect Xtra 5x1m 28 4082 58 i HiTrap Desalting 5x5m 17 1408 01 Troubleshooting ese 11000279 High backpressure HiPrep 26 10 Desalting 1 53 ml 17 5087 01 e Column clogged Clean the column according 4 53 ml 17 5087 02 to instructions Make sure the sample has been Superloop 10 ml 1 18 1113 83 centrifuged and or filtered through a 0 45 um filter 3 e System clogged Replace the column with a piece Superloop 50 ml 1 8 1113 84 of tubing Check pressure If backpressure gt 0 3 MPa Superloop 150 ml 1 18 1023 85 clean system according to manual Pack size available by special order No binding e Check that the correct column is used Check that the inlet tubing from each buffer is connected to the correct inlet port e Check that the composition and pH of the buffers are correct Check that the sample has been adjusted to binding buffer conditions e Check that your sample contains target protein No elution Check that the inlet tubing from each buffer is connected to the correct inlet port e Check that the composition and pH of the buffers are correct e Use alternative elution conditions according to the column instructions e Check that your sample contains target protein 11 0027 48 AC 2007 09 e p23 MAD purification gradient elution 1
39. high purity water and chemicals e Filter all buffers through a 0 45 um filter before use Binding buffer port A1 20 mM sodium phosphate 0 15 M NaCl pH 7 3 Elution buffer port B 50 mM Tris HCl 10 mM reduced glutathione pH 8 0 Prepare at least 500 ml of each eluent 2 Preparing the sample a Adjust the sample to composition of binding buffer by e diluting the sample in binding buffer or e by buffer exchange using HiTrap Desalting or HiPrep 26 10 Desalting b Pass the sample through a 0 45 um filter 3 Preparing the system a Place the inlet tubing from port A1 8 port valve in the binding buffer and the tubing from port B 2 port valve in the elution buffer b Place the three brown waste tubings in waste c Connect the column between port 1 on the injection valve 7 port valve and the UV flow cell see Ordering information on next page for suitable columns d Fill the fraction collector rack with 18 mm tubes minimum 10 and position the white plate on the fractionation arm against the first tube e Connect a sample loop large enough for your sample between port 2 and 6 on the injection valve Use a syringe to manually fill the loop Note If a Superloop is needed additional information is supplied in the instructions for Superloop 11 0027 48 AC 2007 09 e p16 4 Selecting Application Template and starting the method a Check the communication to PrimeView At the lower right corner of
40. ions e Check that your sample contains target protein 11 0027 48 AC 2007 09 e p25 IgM purification 1 Preparing the buffers e Use high purity water and chemicals Filter all buffers through a 0 45 um filter before use Binding buffer port A1 20 mM sodium phosphate 0 8 M NH SO pH 7 5 Elution buffer 1 port B 20 mM sodium phosphate pH 7 5 Regeneration buffer 2 port A2 20 mM sodium phosphate 30 isopropanol pH 7 5 Prepare at least 500 ml of each eluent 2 Preparing the sample e diluting the sample in binding buffer or by buffer exchange using HiTrap Desalting or HiPrep 26 10 Desalting b Pass the sample through a 0 45 um filter 3 Preparing the system a Place each inlet tubing from port A 8 port valve in eluents as given above and the tubing from port B 2 port valve in elution buffer 1 b Place the three brown waste tubings in waste c Connect the column between port 1 on the injection valve 7 port valve and the UV flow cell see Ordering information on next page for suitable columns d Fill the fraction collector rack with 18 mm tubes minimum 15 and position the white plate on the fractionation arm against the first tube e Connect a sample loop large enough for your sample between port 2 and 6 on the injection valve Use a syringe to manually fill the loop Note If a Superloop is needed additional information is supplied in the instructions for Superl
41. ions Make sure the sample has been centrifuged and or filtered through a 0 45 um filter For unclarified samples using HisTrap FF crude make sure the sample has been lysed properly e g using thorough sonication and DNase treatment System clogged Replace the column with a piece of tubing Check pressure If backpressure gt 0 3 MPa clean system according to manual No binding Check that the correct column is used e Check that the inlet tubing from each buffer is connected to the correct inlet port e Check that the composition and pH of the buffers are correct e Check that the sample has been adjusted to binding buffer conditions Check that your sample contains target protein No elution e Check that the inlet tubing from each buffer is connected to the correct inlet port e Check that the composition and pH of the buffers are correct e Use alternative elution conditions according to the column instructions e Check that your sample contains target protein 11 0027 48 AC 2007 09 p9 Histidine tagged protein purification gradient elution 1 Preparing the buffers e Use high purity water and chemicals Filter all buffers through a 0 45 um filter before use Binding buffer port A1 20 mM sodium phosphate 0 5 M NaCl 20 mM imidazole pH 7 4 Elution buffer port B 20 mM sodium phosphate 0 5 M NaCl 0 5 M imidazole pH 7 4 Prepare at least 500 ml of each eluent Alterna
42. ition b Check that the UV lamp is on Set Parameters Lamp on 11 0027 48 AC 2007 09 e p2 Setup and Calibration b Cc d Lock knob Fill the fraction collector with tubes Adjust the height of the delivery arm so that the horizontal mark on the tube sensor is at the same level as the top of the collection tubes Sensor Control Check that the sensor is in the correct position for the tube size The eluent tubing should be positioned above the center of the collection tube Use the red sensor Si control to position the tube holder See each cue card for information on the number of tubes to be used e Tubing holder ___ f g h a b Place the tubing holder over the length guide small hole in the delivery arm push the tubing down to the bottom of the guide and tighten the nut This ensures that the correct length of the tubing is exposed Note Make sure that the end of the tubing is cut leaving a straight edge For more information see AkTAprime plus User Manual Re install the tubing holder into the delivery arm Rotate the rack by hand until the rear half of the tube sensor rests against tube 1 Press feed tube on the front panel The bowl moves to the correct position to collect the first fraction in tube Make sure that drop synchronization is activated Set Parameters
43. mplate T Press OK to start M Y Affinity Purification any HiTrap Run data displayed Theoretical gradient in Affinity Purification any HiTrap Application Template B 100 Elution Priming Equilibration Water wash amp 50 4 priming Sample Re equili 1 10 20 10 5 Min Total separation time 47 min sample application time 5 Typical result Sampl le Human plasma Column HiTrap Benzamidine FF high sub 1 ml Bindin Elution bui AUz80nm LU 15 1 0 0 5 g buffer port A1 50 mM Tris HCl 0 5 M NaCl pH 7 4 fer port B 50 mM Glycine HCI pH 3 0 Human plasma without trypsin like serine proteases 6 UV 280 nm pH ZI a Eluted trypsin like serine proteases Troubleshooting High backpressure Column clogged Clean the column according to instructions Make sure the sample has been centrifuged and or filtered through a 0 45 um filter System clogged Replace the column with a piece of tubing Check pressure If backpressure gt 0 3 MPa clean system according to manual No binding No elutio Check Check ha ha the correct column is used the inlet tubing from each buffer is connected to the correct inlet port Check correct Check ha ha buffer con Check Check ha n ha the composition and pH of the buffers are the sample has been adjusted to binding ditions y
44. n collector with 20 10 30 tubes However note that the maximum capacity of the fraction collector is 95 tubes limiting the sample volume to 75 ml 11 0027 48 AC 2007 09 e p20 4 Selecting Application Template and a b starting the method Check the communication to PrimeView At the lower right corner of the screen the text Controlled By prime should be displayed Use the arrow and OK buttons to move in the menu tree until you find Affinity Purification any HiTrap Set Sample Inj Vol Templates 00 0 ml 00 0 Y y Y Application Template Run Application Template T Press OK to start M Y Affinity Purification any HiTrap Run data displayed c Enter the sample volume and press OK to start the template Note If a 5 ml column is preferred see cue card on p 36 Theoretical gradient in Affinity Purification any HiTrap Application Template 100 50 priming B Elution Priming Equilibration Water wash amp Sample Re equili 1 10 20 10 5 Min Total separation time 47 min sample application time 5 Typical result Sample Clarified lysate of E coli expressing MBP tagged protein Column MBPTrap HP 1 ml Binding buffer port A1 20 mM Tris HCl 200 mM NaCl 1 mM EDTA 1 mM OTT H 7 4 20 mM Tris HCl 200 mM NaCl 1 mM EDTA 1 mM OTT 10 mM maltose pH 7 4 Elution buffer port B AU 280 nm
45. ons to move in the menu tree until you find Albumin Removal HiTrap Blue Set Sample Inj Vol Templates 00 0 ml 00 0 y Y Run Application Template Application Template 7 Press OK to start M Albumin Removal HiTrap Blue Run data displayed Cc Enter the sample volume and press OK to start the template Note If a 5 ml column is preferred see cue card on p 36 Theoretical gradient in Albumin Removal HiTrap Blue Application Template 100 50 B Wash Priming amp Equilibration Sample Re equilibration 11 10 11 6 Min Total separation time 37 min sample application time 5 Typical result Ordering information Sample Human plasma buffer exchanged to binding buffer Product Quantity Code No using HiTrap Desalting __ T Tr rr___t r_ Column HiTrap Blue HP 1 ml HiTrap Blue HP 5x1ml 17 0412 01 Binding buffer A1 20 mM sodium phosphate pH 7 0 3 Elution buffer B 20 mM sodium phosphate 2 0 M NaCl pH 7 0 HiTrap Desalting 5x5ml 17 1408 01 AUze0 nm mS cm 100 x 5 ml 11 0003 29 UV 280 nm HiPrep 26 10 Desalting 1 53 ml 17 5087 01 204 Conductivity x 5 4 53 ml 7 5087 02 Superloop 10 ml x 18 1113 83 Human e CT 150 Superloop 50 ml 1 18 1113 84 Superloop 150 ml 1 18 1023 85 10 100 Albumin free human plasma 27 50 k Up T T T T 15 20 25 30 min Pack size available by special order Tr
46. oop 11 0027 48 AC 2007 09 e p26 4 Selecting Application Template and starting the method Check the communication to PrimeView At the lower right corner of the screen the text Controlled By prime should be displayed a b Use the arrow and OK buttons to move in the menu tree until you find IgM Purification HiTrap IgM Purification Set Sample Inj Vol Templates gt 00 0 ml 00 0 y Y Application Template Run Application Template a Adjust the sample to composition of binding buffer by Press OK to start Y M IgM Purification HiTrap IgM Purification Run data displayed c Enter the sample volume and press OK to start the template Theoretical gradient in IgM Purification HiTrap IgM Purification Application Template B Elution Regeneration 100 7 Priming amp Equilibration 50 Sample Wash Re equilibration Total separation time 48 min sample application time 5 Typical result Sample Hybridoma cell culture containing IgM Column HiTrap IgM Purification HP 1 ml Binding buffer A1 20 mM sodium phosphate 0 8 M NH4 2SO4 pH 7 5 Elution buffer 1 B 20 mM sodium phosphate pH 7 5 Elution buffer 2 A2 20 mM sodium phosphate 30 isopropanol pH 7 5 AU2e0 nm mS cm 2 0 gt m 150 UV 280 nm Conductivity m 120 Ir Mouse IgM Si 90 10 m 60 30 0 n 0 4h T T T T T T Troubleshooting
47. op 150 ml 1 18 1023 85 Troubles hooting Pack size available by special order High backpressure e Column clogged Clean the column according to instructions Make sure the sample has been centrifuged and or filtered through a 0 45 um filter e System clogged Replace the column with a piece of tubing Check pressure If backpressure gt 0 3 MPa clean system according to manual No binding e Check that the correct column is used Check that the inlet tubing from each buffer is connected to the correct inlet port e Check that the composition and pH of the buffers are correct If the compostion and pH of the buffers are correct but there is still no binding a If the protein of interest does not bind to the column the pH should be decreased b If it still not binds it is advisable to try an anion exchanger see cue card Anion exchange c If the protein of interest binds to the column but the separation is poor the pH should be increased e Check that the sample has been adjusted to start buffer conditions e Check that your sample contains target protein No elution Check that the inlet tubing from each buffer is connected to the correct inlet port e Check that the composition and pH of the buffers are correct e Use alternative elution conditions according to the column instructions e Check that your sample contains target protein 11 0027 48 AC 2007 09 e p35 Method Templates value table
48. oubleshooting High backpressure e Column clogged Clean the column according to instructions Make sure the sample has been centrifuged and or filtered through a 0 45 um filter System clogged Replace the column with a piece of tubing Check pressure If backpressure gt 0 3 MPa clean system according to manual No binding e Check that the correct column is used e Check that the inlet tubing from each buffer is connected to the correct inlet port e Check that the composition and pH of the buffers are correct e Check that the sample has been adjusted to binding buffer conditions e Check that your sample contains target protein No elution e Check that the inlet tubing from each buffer is connected to the correct inlet port e Check that the composition and pH of the buffers are correct e Use alternative elution conditions according to the column instructions e Check that your sample contains target protein 11 0027 48 AC 2007 09 e p29 Removal of trypsin like serine proteases 1 Preparing the buffers e Use high purity water and chemicals e Filter all buffers through a 0 45 um filter before use Binding buffer port A1 50 mM Tris HCl 0 5 M NaCl pH 7 4 Elution buffer port B 50 mM glycine HCl buffer pH 3 0 Prepare at least 500 ml of each eluent 2 Preparing the sample a Adjust the sample to composition of binding buffer by e diluting the sample in binding buffer or by
49. our sample contains target protein the inlet tubing from each buffer is connected to the correct inlet port Check correct Use al ha erm the composition and pH of the buffers are ative elution conditions according to the column instructions Check ha your sample contains target protein Ordering information Product HiTrap Benzamidine FF high sub Superloop 10 ml Superloop 50 ml Superloop 150 ml Quantity Code No 2x1ml 17 5143 02 5x1ml 17 5143 01 1 18 1113 83 1 18 1113 84 1 18 1023 85 11 0027 48 AC 2007 09 e p31 Anion exchange 1 Preparing the buffers e Use high purity water and chemicals Filter all buffers through a 0 45 um filter before use Start buffer port A1 20 mM Tris HCl pH 8 0 Elution buffer port B 20 mM Tris HCl 1 0 M NaCl pH 8 0 Prepare at least 500 ml of each buffer Alternative buffers Start buffer port A1 20 mM Glycin NaOH pH 9 5 Elution buffer port B 20 mM Glycin NaOH 1 0 M NaCl pH 9 5 Start buffer port A1 20 mM bis Tris pH 6 5 Elution buffer port B 20 mM bis Tris 1 0 M NaCl pH 6 5 2 Preparing the sample a Adjust the sample to composition of start buffer by e diluting the sample in binding buffer or e by buffer exchange using HiTrap Desalting or HiPrep 26 10 Desalting b Pass the sample through a 0 45 um filter 3 Preparing the system a Place the inlet tubing from port A1 8 port valve in the binding bu
50. ower right corner of the screen the text Controlled By prime should be displayed b Use the arrow and OK buttons to move in the menu tree until you find Desalting HiTrap Desalting Set Sample Inj Vol Templates gt 00 0 ml 00 0 ad v Application Template Run Application Template 1 Press OK to start Y M Desalting HiTrap Desalting Run data displayed 11 0027 48 AC 2007 08 e p4 c Enter the sample volume and press OK to start the template Theoretical gradient in Desalting HiTrap Desalting Application Template B 100 Priming amp Equilibration 50 Elution Sample 6 3 Min Total separation time 9 min sample application time 5 Typical result AUzgonm UV 280 nm Conductivity 0 15 Sample Histidine tagged protein in 20 mM sodium phosphate 0 5 M sodium chloride 0 5 M imidazole pH 7 4 Colum HiTrap Desalting 5 ml Buffer AL 20 mM sodium phosphate 0 15 M sodium chloride pH 7 0 Histidine tagged protein 0 10 Troubleshooting Ordering information High backpressure Product Quantity Code No e Column clogged Clean the column according HiTrap Desalting 5x5 ml 3 17 1408 01 to instructions Make sure the sample has been 100 x 5 ml 11 0003 29 centrifuged and or filtered through a 0 45 um filter Superloop 10 ml x 18 1113 83 System clogged Replace the column with a piece of tubing Check pressure If backpressur
51. p is needed additional information is supplied in the instructions for Superloop 11 0027 48 AC 2007 09 e p24 4 Selecting Application Template and starting the method a Check the communication to PrimeView At the lower right corner of the screen the text Controlled By prime should be displayed b Use the arrow and OK buttons to move in the menu tree until you find Mab Purification Gradient Elution Set Sample Inj Vol Templates gt 00 0 ml 00 0 y Y Application Template Run Application Template T Press OK to start y v Mab Purification Gradient Elution Run data displayed c Enter the sample volume and press OK to start the template Note If a 5 ml column is preferred see cue card on p 36 Theoretical gradient in Mab Purification Gradient Elution Application Template B Wash 2 100 Priming amp Equilibration Elution 50 Sample Re equilibration 11 10 20 17 5 Min Total separation time 63 min sample application time 5 Typical result Ordering information Product Quantity Code No Sample Cell culture supernatant containing mouse IgG2a Column HiTrap Protein A HP 1 ml HiTrap Protein G HP 2x1m 17 0404 03 Binding buffer A1 0 1 M sodium phosphate 0 1 M sodium citrate pH 7 0 a Elution buffer B 0 1 M sodium phosphate 0 1 M sodium citrate pH 3 0 5x1m 7 0404 01 AU280 nm pH HiTrap Protein
52. ple Re equili bration 616 10 20 20 17 5 Min Total separation time 87 min sample application time 5 Typical result Ordering information Sample Clarified homogenate of E coli Product Quantity Code No expressing histidine tagged protein PP__ l Column HiTrap Chelating HP 1 ml HiTrap IMAC HP 5x1ml 17 0920 03 Binding buffer port A1 20 mM phosphate 0 5 M NaCl 20 MM imidazole pH 7 4 HiTrap IMAC FF 5x1ml 17 0921 02 Wash eluent port A2 Distilled water Metal loading eluent port A3 100 mM ZnCl in distilled water HiTrap Chelating HP 5x1ml 17 0408 01 Elution buffer port B 20 mM phosphate 0 5 M NaCl 5 x 0 5 M imidazole pH 7 4 HiTrap Desalting 5x5ml 17 1408 01 sr 8 100x5m 11 0003 29 UV 280 nm A x Programmed B HiPrep 26 10 Desalting 1 53 ml 17 5087 01 4 53 ml 17 5087 02 Superloop 10 ml Il 18 1113 83 Superloop 50 ml 1 18 1113 84 Superloop 150 ml 1 18 1023 85 Pack size available by special order 10 15 20 25 30 35 40 45 50 min Troubleshooting High backpressure e Column clogged Clean the column according to instructions Make sure the sample has been centrifuged and or filtered through a 0 45 um filter e System clogged Replace the column with a piece of tubing Check pressure If backpressure gt 0 3 MPa clean system according to manual No binding e Check that the correct column is used e Check that the inlet tubing from each buffer is conn
53. s OK e For long term storage of the system repeat step b with 20 ethanol down the system Shu 11 0027 48 AC 2007 09 e p3 Buffer exchange on HiTrap Desalting 1 Preparing the buffers e Use high purity water and chemicals e Filter all buffers through a 0 45 um filter before use Buffer port A1 20 mM sodium phosphate 0 15 M NaCl pH 7 0 Prepare at least 500 ml eluent When performing buffer exchange use the appropriate buffer 2 Preparing the sample Pass the sample through a 0 45 um filter The maximum recommended sample volume is 1 5 ml 3 Preparing the system a Place the inlet tubing from port A1 8 port valve and port B 2 port valve in the buffer b Place the three brown waste tubings in waste c Connect the column between port 1 on the injection valve 7 port valve and the UV flow cell see Ordering information on next page for suitable columns d Fill the fraction collector rack with 18 mm tubes minimum 20 and position the white plate on the fractionation arm against the first tube e Connect a sample loop large enough for your sample between port 2 and 6 on the injection valve Use a syringe to manually fill the loop Note If the same sample is applied repeatedly a Superloop can be used For information of how to use it see the instructions for the Superloop 4 Selecting Application Template and starting the method a Check the communication to PrimeView At the l
54. st published Feb 2005 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 request Contact your local GE Healthcare representative for the most current information GE Healthcare UK Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK GE Healthcare Europe GmbH Munzinger Strasse 5 D 79111 Freiburg Germany 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 11 0027 48 AC 12 2007
55. t the correct column is used e Check that the inlet tubing from each buffer is connected to the correct inlet port e Check that the composition and pH of the buffers are correct If the compostion and pH of the buffers are correct but there is still no binding a If the protein of interest does not bind to the column the pH should be increased b If it still not binds it is advisable to try a cation exchanger see cue card Cation exchange c If the protein of interest binds to the column but the separation is poor the pH should be decreased e Check that the sample has been adjusted to start buffer conditions Check that your sample contains target protein No elution e Check that the inlet tubing from each buffer is connected to the correct inlet port e Check that the composition and pH of the buffers are correct e Use alternative elution conditions according to the column instructions Check that your sample contains target protein 11 0027 48 AC 2007 09 e p33 Cation exchange 1 Preparing the buffers e Use high purity water and chemicals Filter all buffers thtough a 0 45 um filter before use Start buffer port A1 50 mM sodium acetate pH 5 5 Elution buffer port B 50 mM sodium acetate 1 0 M NaCl pH 5 5 Prepare at least 500 ml of each eluent Alternative buffers Start buffer port A1 20 mM sodium phosphate pH 7 0 Elution buffer port B 20 mM sodium phosphate
56. ta displayed c Enter the sample volume and press OK to start the template Note If a 5 ml column is preferred see cue card on p 36 Theoretical gradient in Affinity Purification any HiTrap Application Template 100 50 priming B Elution Priming Equilibration Water wash amp Re equili bration 1 10 20 10 5 Min Total separation time 47 min sample application time 5 Typical result Ordering information Sample Clarified homogenate of E coli expressing histidine tagged Product Quantity Code No tei sa ff aaa AI CORTA Column Histropm HPiml HisTrap HP Sx1m 7 5247 01 Binding buffer port A1 20 mM phosphate 0 5 M NaCl 20 mM 100 x 1 mi 17 5247 05 imidazole pH 7 4 Hi 7 Bi Elution buffer port B 20 mM phosphate 0 5 M NaCl 0 5 M isTrap FF Sx1m 7 319 01 imidazole pH 7 4 100 x 1 ml 17 5319 02 AUzg0nm B a HisTrap FF crude 5x1m 11 0004 58 100 UV 280 nm 100 x 1 mI 11 0004 59 254 Programmed 8 g BS HiTrap Desalting 5x5m 17 1408 01 20 100 x 5 ml 11 0003 29 L 60 HiPrep 26 10 Desalting 1 53 ml 17 5087 01 154 4 53 ml 17 5087 02 L 40 Superloop 10 ml 1 18 1113 83 105 Superloop 50 ml 1 18 1113 84 0 54 M 20 Superloop 150 ml 1 18 1023 85 04 L o Pack size available by special order T T T T T T 5 10 15 20 25 30 35 min Troubleshooting High backpressure e Column clogged Clean the column according to instruct
57. the screen the text Controlled By prime should be displayed b Use the arrow and OK buttons to move in the menu tree until you find GST tag Purification GSTrap Toon SE Y Application Template Run Application Template T Press OK to start Vv M este FALSO Run data displayed c Enter the sample volume and press OK to start the template Note If a 5 ml column is preferred see cue card on p 36 Theoretical gradient in GST tag Purification GSTrap Application Template Elution B 100 Priming amp Equilibration 50 Sample Re equilibration 11 10 11 6 Min Total separation time 37 min sample application time 5 Typical result Ordering information Sample Clarified homogenate of E coli expressing Product Quantity Code No GST tagged protein 7 Column GSTrap FF 1 ml GSTrap FF 2x1m 7 5130 02 Binding buffer A1 20 mM sodium phosphate 0 15 M NaCl pH 7 3 17 5 Elution buffer B 50 mM Tris HCl 10 mM reduced glutathione pH 8 0 sx1m 7 5130 01 AU2500m B 00x1m 7 5130 05 UV 280 4 Pidgrommed B GSTrap HP 5x1m 7 5281 01 164 ai 100x1m 17 5281 05 y 80 GSTrap 48 5x1m 28 4017 45 100x 1ml 28 4017 46 nee HiTrap Desalting 5x5m 17 1408 01 0 8 4 SHE 100 x 5 ml 11 0003 29 agged L 40 proren HiPrep 26 10 Desalting 1153 ml 17 5087 01 F Y 4 53 ml 17 5087 02 Inject m 20 J N Superloop 10 ml 1 18 1
58. tive binding buffers 5 40 mM imidazole can be included in the binding buffer to reduce unspecific binding of non histidine tagged proteins The concentration of imidazole is protein dependent and if the protein of interest elutes or does not bind at a certain imidazole concentration then reduce the concentration 2 Preparing the sample a Adjust the sample to composition of binding buffer by e diluting the sample in binding buffer or e by buffer exchange using HiTrap Desalting or HiPrep 26 10 Desalting b Pass the sample through a 0 45 um filter Note If HisTrap FF crude column is used nor filtration nor clarification of the sample is needed 3 Preparing the system a Place the inlet tubing from port A1 8 port valve in the binding buffer and the tubing from port B 2 port valve in the elution buffer b Place the three brown waste tubings in waste c Connect the column between port 1 on the injection valve 7 port valve and the UV flow cell see Ordering information on next page for suitable columns d Fill the fraction collector rack with 18 mm tubes minimum 40 and position the white plate on the fractionation arm against the first tube e Connect a sample loop large enough for your sample between port 2 and 6 on the injection valve Use a syringe to manually fill the loop Note If a Superloop is needed additional information is supplied in the instructions for Superloop 11 0027 48 AC 2007 09 e p10
59. tree until you find On Column Refolding HisTrap Set Sample Inj Vol Templates gt 00 0 mb 60 0 y Y Application Template Run Application Template Press OK to start M Y On Column Refolding HisTrap Run data displayed c Enter the sample volume and press OK to start the template Note If a 5 ml column is preferred see cue card on p 36 Theoretical gradient in On column Refolding HisTrap Application Template B 100 7 Refolding Elution Priming Buffer wash 50 7 Equili amp Priming bration Re equilibration Sample gt 210 30 60 20 20 17 Min Total separation time 160 min sample application time 5 Typical result Sample Clarified homogenate of E coli expressing histidine tagged protein Column HisTrap FF 1 ml Binding buffer A1 6 M Guanidine hydrochloride 20 mM Tris HCl 0 5 M NaCl 5 mM Imidazole 1mM 2 mercaptoethanol pH 8 0 Solubilization buffer port A2 6 M Urea 20 mM Tris HCl 0 5 M NaCl 5 mM Imidazole 1 mM 2 mercaptoethanol pH 8 0 20 mM Tris HCl 0 5 M NaCl 0 5 M Imidazole 1 mM 2 mercaptoethanol pH 8 0 20 mM Tris HCl 0 5 M NaCl 5 mM Imidazole 1 mM 2 mercaptoethanol pH 8 0 Elution buffer port A3 Refolding buffer port B AU280 nm B UV 280 nm 100 Programmed B 0 44 80 0 354 60 0 2 4 40 0 14 20 0 0 20 40 60 80 100 120 140 min Troubleshooting High backpressure
60. valve Use a syringe to manually fill the loop Note If a Superloop is needed additional information is supplied in the instructions for Superloop 11 0027 48 AC 2007 09 e p22 4 Selecting Application Template and a b starting the method Check the communication to PrimeView At the lower right corner of the screen the text Controlled By prime should be displayed Use the arrow and OK buttons to move in the menu tree until you find Mab Purification Step Elution Set Sample Inj Vol Templates 00 0 ml 00 0 y Y Run Application Template Application Template T Press OK to start Y Mab Purification Step Elution Run data displayed c Enter the sample volume and press OK to start the template Note If a 5 ml column is preferred see cue card on p 36 Theoretical gradient in Mab Purification Step Elution Application Template 100 7 50 Elution B Priming amp Equilibration Sample Re equilibration 11 10 11 6 Min Total separation time 37 min sample application time 5 Typical result Ordering information AUzzonm PH san ain Product Quantity Code No Zw a tr eis P HiTrap Protein G HP 2x1m 17 0404 03 5x1m 17 0404 01 See aint Pace HiTrap Protein A HP 2x1m 17 0402 03 Column HiTrap Protein G HP 1 ml Binding buffer A1 20 mM sodium 5x1im 17 0402 01 phosphate pH 7 0 Elution buffer B 0 1 M

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