I have been having an issue with my western blots where the bands appear at a completely different size. I have probed for these proteins in the past with success and the same samples have been probed by another colleague with no issues.
These are all phosphorylated proteins and we use a pre-cast Nupage Bis-Tris gel 4-12% with MES running buffer. The ponceau stain (attached) looks fine too.
HELP!
Vaibhav,
Phosporylated proteins (esp. with P-Tyr) migrate differently, as they bind different amount of SDS, compared to the unphosphorylated variety.
Is your gel system the same as the others you are referring to?
Have you spotted the negative signal (white bands)? This could be due to depletion of the ECL reagent due to very high peroxidase activity, so all the substrate has been used up before you could get the blot into the imager.
The protein that I am probing for (and have probed for before multiple times) is at 42/44. Using the exact same protocol, I have had no issues with detection. I have not seen white bands, and just re-ran new samples with the same result...
Why you didn't loaded a negative signal bands? That it could be due to depleted the ECL solution , so all the substrate has been used up before you could get the blot into the imager.
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