I am working on expression and purification of one cytoplasmic protein with His tag, in E coli Bl21(DE3) host cell. Here is SDS picture. Line 2 and 9 are cell lysate, 3,4,7,8 wash steps and 5,6 are elutes using different purification procedure. The expected size is 48 kDa. For protein extraction I used a high pressure homogenizer, also I didn’t use any inhibitors. I was told to try to use Bugbuster protein extraction reagent supplemented with benzonase, do you think it might help?
Proteins don't always run at the expected molecular weight on SDS-PAGE. I'd suggest sending a sample of the mostly purified protein off to a mass spec lab to determine the molecular weight of the whole protein.
Meanwhile, it wouldn't hurt to include protease inhibitors when you lyse the cells. There might be a protease-sensitive site near the N- or C-terminus.
I agree with Adam B Shapiro , run LC-MS to confirm the theoretical size with the purified material. The protease inhibitor additional is a solid addition. Also, not sure what your wash conditions are for your Ni-NTA column but I would either increase the wash volume or imidazole concentration to reach better first pass purity. You may also want to run SEC to polish the material.
Alexey Tarabarov
it is not always straightforward to determine MW of 50kDa protein. You will also lose a lot of material while transferring protein from the SDS gel. I would recommend to perform in-gel digestion followed by LCMS on the peptides. You have enough material to get a good peptide map coverage and speculate either you have any C-terminal or N-terminal truncations. If the peptide map shows nearly a full protein coverage, our protein just migrates a bit lower on the SDS gel. good luck
Adam B Shapiro Thomas J Esparza Alexandre Podtelejnikov Thank you everyone for your suggestions. Somehow the problem solved when I added 10% glycerol to the elution buffer.
In that case, the likely scenario was that the protein underwent a site-specific proteolysis due to instability exposing a protease-sensitive site. The glycerol stabilized the protein, preventing the proteolysis.
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