I'm trying to express a His-tagged protein in BL21 (E.coli) cells. Until recently, in order to lyse the cells, I would always use sonication. I switched to freeze-thaw, as efficiency was better compared to physical disruption of the cells. However, the lysate from freeze-thaw is very viscous. Even after spinning for 1.5 hours at 15000rcf, the supernatant is still viscous. Loading this supernatant, appears to slow down the purification (the drain from the column is dead slow). We do not have DNAase in our lab. Kindly provide your inputs, on any other strategies that could help troubleshoot this problem. Thanks.
Try to dilute the lysate with GST buffer or whichever buffer you are using to purify the protein!! For my purification i do sonication followed by triton X 100 treatment for 1 hour then i clear the lysate using centrifuge. It is working fine.
Oscar is right, gDNA is the reason for high viscosity. Another suggestion beforeyou centrifuge, is to treat your lysate with Dnase I briefly (5-10 min incubation should do the trick).
After lysis we generally spin down at 18000rpm for half an hour and we get clear supernatant. Try centrifuging longer.
hello Srikantha,
Viscosity of the sample results from release of nucleic acids and these can easily be removed by adding cationic compounds like streptomycin sulfate or protamine sulfate which will precipiate gDNA. An alternate approach would be to do differential ammonium sulfate precipitation which will result in higher fold purification as well apart from getting rid of gDNA .
Best of Luck
After digestion with DNAse or DNA release, I have tried filtering the lysate with a 0.45uM and 0.22uM filter. It is a very labor intensive but it works well.
Don't forget Rnase treatment to rid the lysate of RNA. There is likely much more RNA than DNA in your lysate, but you should treat with both RNAse and DNase.
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