I ran a western blot using 12% gel and probing for ERK/pERK (cell signaling antibodies). The green is total ERK, the Red is supposed to be phospho-ERK. I see 2 bands below the total ERK that express the red pERK antibody... but this shouldn't be there.. the phosphorylated protein should be around the same size or higher than the non-phosphorylated ERK... anybody else have this problem? What are these bands?
Hi there,
Probably degradation fragments of pERK bearing the phosphorylation.
Phospho ERK (MAP Kinase) runs faster than the non Phosphorylated form due to the negative charge from the phosphate. The way your gel ran is an artifact of the gel. Check the gel expiration and your buffers. There should be one green band above one red band. BTW... I have in my past performed many map kinase gels.
Disagree with Tony: pERK does not run faster. Impact of phosphorylation on SDS-PAGE is rather unpredictable, but often makes things runs slower in my experience. For ERK, the overlay is pretty good (for p202/204 at least, in my experience). As for the highlighted red bands, likely non-specific (not related to ERK), but hard to tell unless other controls are performed (siRNA etc). Tony has a point though: you should be able to see some yellow signal in your specific signals from the pX/X overlay. If not, not sure what you are seeing at 42/44 kDa. Consider using a positive control (e.g. PMA).
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