I try to purify a specific recombinantly expressed protein, but I am facing some trouble with my lysed cell solution after centrifugation. Normally I would expect to get a clear solution and a nice debris pellet at the bottom of the tube after centrifugation of the lysed cell solution. However, currently I get a very turbid and highly viscous solution. This makes it nearly impossible to sterile filter it afterwards.
Does anyone have an idea why my solution is so viscous or what I can do against it?
Here is in detail what I do:
Cell lysis buffer containing 50 mM NaPO4, 0.5 M NaCl, 10 mM imidazole, 2 mg/ml lysozyme, 1 mM PMSF and 0.1 mg/ml DNAse. After incubating this solution with my cell pellet (4 ml buffer / 1 g cell pellet) for 30 min, I perform a ultrasonication of the cell solution (2x 1 min with 1 min break between).
Now I centrifuge the cell solution for 30 min at 8960 rcf.
All steps are performed on ice, cell line is Shuffle T7 E.coli strain.
I am very thankful for any help.
Dear Pascal
Just some questions:
1) is your sample at the end of the sonication hot?
2) which is the pH of your lysis buffer and the pI of your protein?
3) Did you tried to increase centifugatino speed? or it is the maximun that you can reach in your lab?
In my experience, when i'm you using sonication i resuspend the cells in higher buffer volume (at least 10ml for each g of wet biomass) and i perfoming more sonication cicles but shorter to be sure that the temperature during the sonication do not increase.
best regards
Manuele
DNA is most likely the cause of the high viscosity. You added DNase, but you did not add Mg2+, which is required for DNase activity.
Thanks for all the helpful answers!
Mazin Nadhim Mousa : Could you explain this for me?
Manuele Martinelli : 1) I don't think that it is hot. The Tube (50ml Falcon tube) containing the protein solution is embedded in ice during sonication. So it should be cool. But yes, reducing the sonication time and increasing the cycles might be a good idea.
2) pH of the buffer is 7.5, pI of my protein is 8.95.
3) Our normal centrifuges can't go higher in centrifugation speed, however I could use an ultracentrifuge. I also thought about that option, this might solve the problem. However, some months ago I didn't had these problems. Thats why it's strange that I have to change my protocol now.
Adam B Shapiro : This is a very good point. Do you have a recommodation for the Mg2+ concentration in the solution?
Adam B Shapiro : I did it again with MgCl2 and it worked much better this time. Thank you very much for your help!
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