Dear all,
I have been trying to extract protein from peripheral blood and bone marrow dry pellets.
I have been using RIPA buffer, cOmplete protease inhibitor cocktail (Roche) and PMSF.
When quantifying proteins with Bradford, it detects a protein concentration around 2 ug/ul and beta-actin is visible in Western Blot.
Unfortunately, when trying to detect high molecular weight proteins, no bands are visible (specifically, I am trying to quantify SMARCAL1 protein).
I think I should add or change protease inhibitors to avoid protein loss, but is there someone who could kindly help with experience and advice?
Thank you in advance!
There are 2 major possibilities.
1. the larger proteins were never solubilized and therefore not visible in (I presume) gel electrophoresis. If so, when staining the gel, you may see a stained “band’ at the bottom of the well. This would indicate insoluble proteins are present in the sample and cannot travel in the liquid phase. it is possible that these proteins may have crosslinked and cannot enter the polyacrylamide gel.
2. another possibility is that the gels you are using are not the best for the protein..perhaps you need a smaller % of polyacrylamide or less crosslinker. lower polyacrylamide/crosslinker resolve larger proteins. Again, you may see a stained band at the bottom of the well.
if you can’t see any staining at the bottom of the well, then your protein may be highly insoluble. If so, try to solubilizarme the dry pellets with different detergents (e.g. Triton x-100, SDS, laurel maltonside) preferably a non-ionic detergent (since these are easier to remove later on).
finally, I would recommend you use the BCA protein assay instead of the Bradford since it gives you better results.
good luck
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