Imidazole reduction causes precipitation - protein purification (Jul/14/2005 )
I have extracted a 24kDa protein that is phosphorylated from a Ni column.
I run a gradient from 8M urea to 0. I elute with 500mM imidazole and I have had previous results where I get my specific protein. When I try to get rid of the imidazole the protein crystalizes and I lose about 90%. I was wondering if anyone has any ideas on how to reduce imidazole concentration without the precip?
At what point does it crystallize? Does any dilution of the 500 mM solution result in precipitation?
My initial thought is that you're reducing the ionic strength of the solution too quickly, or perhaps drastically altering the pH.
I would suggest first taking some of your precipitated sample, and add back imidazole to see if it redissolves. If it is reversible, you may be able to do the same thing by adding similar levels of NaCl.
hi.. using imidazole... very hard to get ride of it..
maybe u can try adjusting the buffer ph to elute ..
or another suggestion would be using L-histidine.. this is the better way i'll be using