Size Exclusion Column - Proteins keep sticking!! - (Apr/28/2005 )
I'm doing work in a new crystalography lab and we have been preparing our proteins on an Akta Purifier first using a His-tag Ni-ATA media, then a TMAE ion exchange column, and last an s200 size exclusion column.
The problem I seem to be having is that as the protein comes off of the size exclusion, it seems to be sticking to itself causing a wide double peak. It has been doing this for a number of different proteins, not just one type.
The proteins are soluble on induction checks, I'm adding bME to the lysis buffer. Just wondering if there is something I am missing to try to get these proteins unstuck!
Any help would be most welcome.
The problem I seem to be having is that as the protein comes off of the size exclusion, it seems to be sticking to itself causing a wide double peak. It has been doing this for a number of different proteins, not just one type.
The proteins are soluble on induction checks, I'm adding bME to the lysis buffer. Just wondering if there is something I am missing to try to get these proteins unstuck!
Any help would be most welcome.
Greetings Jedi!
Gel filtration shouldn't cause the problems you described. Might be possible to get proteins aggregated when you concentrate, but gel filtration does the exact opposite... Try to increase the running buffers salt concentration (you probably have at the moment ~150 mM NaCl)? Try changing the buffering agent, phosphate buffers can cause "sticking" of proteins. Try using Tris or Hepes buffer instead (or any other suitable to your protein). Try adding detergents Triton, Tween or CHAPS. If none of these helps run in high UREA concentration
Ben
Thank you Obi-wan kenobi - you are my only hope
I've added triton to the lysis buffer and the NaCL conc. is at 150mM. I will consider those other buffers as well.
Talknig to the PI, it seems that it is just a product of the surface charges of the proteins. Oh well, I'll keep at it.
Thanks!