While running gels for western blotting, I am currently getting, what seems to be, the blue dye from my sample buffer migrating more slowly in sample lanes leaving blue streaks. These transfer to the membrane and show up in my 700 and 800 channels (700 is attached). I can also see it has affected the migration of the proteins in my (weak) ponceau stain as well.
The samples being run are cell lysates from a photocrosslinking experiment with a site-specific crosslinker, pBpa. The lysis was done in a buffer that's 1% Triton X-100 and contains 1 mM DTT.
The gels are Thermo's NuPAGE 4 to 12%, Bis-Tris, and they're being run in the NuPAGE MOPS buffer.
If anyone has advice on how to go about troubleshooting this, that would be great!
In my opinion, a rest of organic solvents in the loaded sample may cause such a diffuse band/pattern .Another explanation may be an incorrect composition of buffers for the gel and/or runnig buffer.
This happens for several reasons prime reason is like what Andreas Eisenreich stated the presence of organic/lipidic matter in sample which is obvious since these are lysates. Another reason is insufficient loading dye and heat treatment to the samples and last reason is presence of high salt in sample. Also use fresh running buffer
In addition to what is said above (with which I fully agree) you most likely have to low pH of your samples. Orange colour of bromophenol is the evidence. Your gel and running buffer are probably alright (since the ladder looks nice), so your samples is culprit. If its a big deal to change the buffer, try increasing the pH: a drop of 1M tris (just base) should do.
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