western blot mystery

criss.hartzell at emory.edu criss.hartzell at emory.edu
Wed Dec 8 09:27:01 EST 2004


Sorry- my email address was not shown on the previous post.

I would appreciate suggestions on how to interpret the following result.
I have SDS extracts from untransfected HEK293 cells and HEK293 cells
transfected with cDNA for a protein called mB2. On westerns with mB2
antibody, there is a nice band at the predicted 57kD in transfected cells
and no band in untransfected cells. So far, it looks clean. However, if I
add the SDS extracts from the untransfected cells to the extract from
transfected cells, the intensity of the mB2 band decreases with increasing
amounts of untransfected extract added (the gel is loaded with the same
amount of transfected protein in each lane - the total amount of protein per
lane is increased by addition of untransfected extract). At a ratio of 1:1,
the mB2 band completely disappears. It is not due to proteolysis because the
extracts have been heated to 100C for 10 min prior to mixing. The mB2 band
is not migrating at a different place in the gel, because no signal is found
either in the stacking gel or elsewhere. Adding BSA or serum to the
transfected cell extract does not have the same effect except at very high
concentrations. Has anyone seen anything like this before?

Criss Hartzell






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