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[Nezura]

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?

Thanks!

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Edited by Nezura, 10 February 2011 - 07:44 PM.

[mdfenko]

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.