Hello,
I have a problem during purifying my two proteins using Ni beads (GE healthcare). These two small proteins (about 6 kDa) were expressed in E. coli with an N-terminal His-SUMO tag. When I loaded cell lysate after centrifugation to Ni beads, the mixture became gel-like. And only part of these proteins could be eluted from beads, and these proteins became gel again. My lysis buffer was 20 mM Hepes pH 7.5, 500 mM NaCl, 5% glycerol, 5 mM beta-ME and 1 mM PMSF. I didn't meet this situation before. Can anyone tell me why this happened and how to deal with this problem? Thanks a million!
Hello Zhubing,
I think you need to add DnaseI to your lysis buffer before break the cells
(DNA can form a gel like structure if your not treating them with DNAases).
If you keep the cell pellet @ -80 long time, you may get a situation like this.
you can even try with fresh cell pellet (without freezing them)
Hope this may help you
Himesh makala
Hi Zhubing, this has happened to me too.
Once i prepared several cell lystae samples, and some of them became turbid when i added them to streptavidin beads.
May be the total protein concentration of the sample was too high as in my case those samples with less cell didn't meet this problem.
Hope you sovle the problem and know the reason.
Similar experience here. Definitely try DNase1 (or longer sonication maybe?), with a large amount of cells chromosomal DNA cause such a problem
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