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ÄKTAprime plus

GE Healthcare

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(II)-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

Содержание AKTAprime plus

Страница 1: ...tein purification gradient elution 10 IMAC purification any metal 12 On column refolding 14 GST tagged protein purification 16 Strep II tagged protein purification 18 MBP tagged protein purification 2...

Страница 2: ...d 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...

Страница 3: ...t 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...

Страница 4: ...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 Sup...

Страница 5: ...der 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 m filter System clogged Replace...

Страница 6: ...l 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...

Страница 7: ...84 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 m filter System clogged Replace...

Страница 8: ...or by buffer exchange using HiTrap Desalting or HiPrep 26 10 Desalting b Pass the sample through a 0 45 m filter Note If HisTrap FF crude column is used no filtration nor clarification of the sample i...

Страница 9: ...ssure 0 3 MPa clean system according to manual No binding Check that the correct column is used Check that the inlet tubing from each buffer is connected to the correct inlet port Check that the compo...

Страница 10: ...m 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 co...

Страница 11: ...piece of tubing Check pressure If backpressure 0 3 MPa clean system according to manual No binding Check that the correct column is used Check that the inlet tubing from each buffer is connected to t...

Страница 12: ...e 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 s...

Страница 13: ...t tubing from each buffer is connected to the correct inlet port Check that the composition and pH of the buffers are correct Check that the sample has been adjusted to binding buffer conditions Check...

Страница 14: ...imidazole is protein dependent and if the protein of interest elutes or does not bind at a certain imidazole concentration then reduce the concentration Include the same imidazole concentration as in...

Страница 15: ...omposition and pH of the buffers are correct Check that the sample has been adjusted to binding buffer conditions Check that your sample contains target protein No elution Check that the inlet tubing...

Страница 16: ...ill 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 po...

Страница 17: ...clean system according to manual No binding Check that the correct column is used Check that the inlet tubing from each buffer is connected to the correct inlet port Check that the composition and pH...

Страница 18: ...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 n...

Страница 19: ...that the correct column is used Check that the inlet tubing from each buffer is connected to the correct inlet port Check that the composition and pH of the buffers are correct Check that the sample...

Страница 20: ...r 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...

Страница 21: ...at the correct column is used Check that the inlet tubing from each buffer is connected to the correct inlet port Check that the composition and pH of the buffers are correct Check that the sample has...

Страница 22: ...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 f...

Страница 23: ...tem clogged Replace the column with a piece of tubing Check pressure If backpressure 0 3 MPa clean system according to manual No binding Check that the correct column is used Check that the inlet tubi...

Страница 24: ...ter 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...

Страница 25: ...n with a piece of tubing Check pressure If backpressure 0 3 MPa clean system according to manual No binding Check that the correct column is used Check that the inlet tubing from each buffer is connec...

Страница 26: ...e 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...

Страница 27: ...ck that the inlet tubing from each buffer is connected to the correct inlet port Check that the composition and pH of the buffers are correct Check that the sample has been adjusted to binding buffer...

Страница 28: ...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...

Страница 29: ...is used Check that the inlet tubing from each buffer is connected to the correct inlet port Check that the composition and pH of the buffers are correct Check that the sample has been adjusted to bin...

Страница 30: ...or 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 inject...

Страница 31: ...om each buffer is connected to the correct inlet port Check that the composition and pH of the buffers are correct Check that the sample has been adjusted to binding buffer conditions Check that your...

Страница 32: ...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 aga...

Страница 33: ...that the inlet tubing from each buffer is connected to the correct inlet port Check that the composition and pH of the buffers are correct If the compostion and pH of the buffers are correct but ther...

Страница 34: ...ve 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 fract...

Страница 35: ...o the correct inlet port 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 doe...

Страница 36: ...17 5111 01 HiTrap IgY Purification 5 ml 0 5 5 2 5 25 50 50 40 17 5247 01 HisTrap HP 1 ml 0 5 1 0 5 5 20 5 0 17 5248 02 HisTrap HP 5 ml 0 5 5 2 5 25 100 25 0 17 5319 01 HisTrap FF 1 ml 0 5 1 0 5 5 20...

Страница 37: ...5 1 1 5 2 20 5 17 5163 01 HiTrap ANX FF high sub 5 ml 0 5 5 5 25 10 100 25 17 5158 01 HiTrap Q XL 1 ml 0 5 1 1 5 2 20 5 17 5159 01 HiTrap Q XL 5 ml 0 5 5 5 25 10 100 25 17 1151 01 HiTrap SP HP 1 ml 0...

Страница 38: ...1 HiPrep 16 10 Phenyl FF low sub 0 5 5 5 100 40 400 100 17 5097 01 HiPrep 16 10 Octyl FF 0 5 5 5 100 40 400 100 17 5096 01 HiPrep 16 10 Butyl FF 0 5 5 5 100 40 400 100 17 1085 01 HiLoad 16 10 Phenyl S...

Страница 39: ...11 0027 48 AC 2007 09 p39...

Страница 40: ...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 purchas...

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