Protein aggregation (HELP!) - (Sep/06/2006 )
Hi everyone, i have a big problem!!!!!
Im purifying a protein with histag under denaturing conditions (urea 8M) and untill here is everithing right. But when I pass the protein sample through a G-25 or PD-10 desalting column my protein aggregates almost instantly. I tried dialisis but didnt worked, and also tried buffers containing arginine and glycerol to stabilze but didnt work also.
Buffer used in the first time: Buffer P 20mM, NaCl 0.2M and DTT 1mM ph 8
I hope anyone can give me some advice to prevent aggregation,and thank you all.
high salt concentration should be lowered as it favours hydrophic interaction which might be a reason for aggregation; 50 mM NaCl should be sufficient to save protein function
Im purifying a protein with histag under denaturing conditions (urea 8M) and untill here is everithing right. But when I pass the protein sample through a G-25 or PD-10 desalting column my protein aggregates almost instantly. I tried dialisis but didnt worked, and also tried buffers containing arginine and glycerol to stabilze but didnt work also.
Buffer used in the first time: Buffer P 20mM, NaCl 0.2M and DTT 1mM ph 8
I hope anyone can give me some advice to prevent aggregation,and thank you all.
thanks for the advice i will try it. If you realize something more please tell me. THANKS
ionic strength is even to lower to about 20 mM; mild addition of detergents, often used triton x-100 (~ 0.01%) or Nonidet P40 (or IPGAL) or CHAPS or Cholat may also help, if they will not disturb f.i. for crystallization; good luck!