Protein is not binding to the ion-exchange column. - (Feb/26/2008 )

Dear all,
I have one protein in 50mM phosphate buffer pH8.0 contains also 300mM salt, 10mM imidazole, 10% Glycerol and betemercaptoethanol. I can easily purify the protein from NiNTA beads (batch top), elute it with .5M imidazole. I am washing the beads very well but it is not pure enough. Anyway I need pure pure protien for NMR and also concentrated. Th theoretical pI is 8.9 (pretty high) according to the literature, my protein is going to be positively charged under its pI and bind to the cation exchange column, right? then I used cationic exchange column at pH 6.0 and nothing came out even for 1M NaCl. then I just washed the column with pH 9.0 and viola...it has came out. Then I thought maybe protein sticks very tightly to the column. Then I load the protein in the cation ex with pH 7.6..then nothing has came out...
SO, can anybody tell me how come I will purify this protein by ion exchange column?
Thank you for your time all in advance,
DBT

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Sometimes the theoretical pI is way out. have your tried loading onto an anion column at pH 8? When you say you washed the column with pH9, what else was in the solution? You may have been lucky that there was plenty of salt to prevent aggregation of the protein (assuming the calculated pI is correct).

You can test both anion and cation media to see the optimal conditions for binding and elution. Take loose media and equilibrate in a range of pHs. Add your sample batch-wise and mix for 10 minutes by gentle inverting. Spin the beads out in a microfuge then remove the supernatant. Run beads and S/N on SDS PAGE (don't forget to boil the beads!), and you'll find the best pH and media for binding and eluting (it will be the lowest pH where all of the protein binds-that is, nothing in the S/N). Next, mix some of your sample with more beads equilibrated at the optimal pH; spin the beads and remove the S/N. Split this into a number of tubes and add some buffer with different amounts of salt. Run again on a gel, and you have the optimal [NaCl] for elution.

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this handbook may help:

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Weird enough!!with that theoretical pI, protein binds to anion exchange column at pH 7.6 and can be eluted ~200mM salt. But then - when it aggregates approx 30 hours later. I think it it due to the hydrophobicity of the protein 37%. how can we prevent the aggregation in this case? detergent? low salt? low pH? i have already 5% glycerol in the solution..
thnx in advance:)

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we have used 1-5% peg-8000 in the buffer to help prevent aggregation of a few of our proteins. the range is because we add a lower amount to our protein (column buffer) and it increases in concentration when we concentrate the protein by ultrafiltration with a cutoff of 10 kDa.

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