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GAPDH merging lanes - (Feb/10/2011 )

Hi there I'm getting bands like this for my GAPDH control and I'm not sure why the bands aren't more defined.

I'm running a 5% stacking and 10% separating gel. Gel is run at 100V for about 2 hours. Transfered to PVDF membrane at 100V overnight wet with ice block (can this be it?). Using a HRP staining system

Another problem I'm having is on the same gel but probed with a different antibody. The protein I'm interested in should just be one band at 76kDa (bottom visible horizontal lane of second gel) but I'm getting a whole bunch of staining on the top. I never used to get this. Could it be because I'm using a new lot of the same antibody from the same manufacturer? Is it non-specific staining or is my protein just getting separately improperly?

Attached Image

Attached Image


for the second question, it looks like the antibody is not as specific as it should be.

for the first question, what do you mean by not well defined? i see them running together, a little. that can happen for a variety of reasons such as: letting the gel sit after running, allowing the proteins to diffuse; equilibrating with transfer buffer a little too long; a little too much protein loaded; a little too much salt in the sample; other additives in the sample; etc.