Hello, I was running a 12% SDS Page electrophoresis on few granulosa cell samples and got this result after the ponceau staining. The total protein lysate seem to aggregate at 70 kDa ladder mark and doesnt travel further down the gel. I isplated the proteins with RIPA and then denaturated (95C, 5minutes) the sample dilution of 1x LD buffer and 1 ul 1M DTT per 25ul of sample. Any help?
Are you sure that you took only your cells and not too much of the culture medium. These bands seem to be full of BSA (from the serum in the medium?), so you don't see the proteins from the cell lysis which would be throughout the whole lane. Did you wash the cells before to get rid of the medium?
Yeah, I think it could be an abundant albumin from the FBS. I did not wash the pellet because I'm new to WB and didnt expect this issue at all. Is it possible that these aggregates block the migration of other smaller proteins? What can I do to solve this problem?
I don't think that the BSA blocks the migration. It appears so strong that the background seems low in cellular proteins. However it could be that your lysis wasn't efficient, which could result in low protein amount in your sample. So, wash your cells well before lysing and check the protein bands again after lysis.
How do I wash the cells? And how to improve the lysis? thank you! Oh and I was asking about the possible blockage of migration because all of my proteins of interest (that should be located lower) stayed at the BSA site.
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