I am doing western blotting of a protein tag with Anti-Myc. It seems that it has some unspecific, smear band both up side and lower side of the target band (please see image below). Can anyone explain why this may have appeared?
Smears in western blots sometimes are the result of too much product being loaded into the gel or In some other cases, when the antibodies are highly specific, do to the glycosylation state of the protein. If your protein of interest is highly glycosylated and you are just looking for expression levels and not post-translational modifications of the protein you can use de-glycosylation kits or compounds to clear up the smear.
As some of the comments above have stated, there is also the possibility that you might be looking at some protein degradation, or an antibody with some degree of non-specificity, in which case you may want to make sure that enough protease and phosphatase inhibitors are present in your buffer. You can also try washing longer with a high salt buffer right after incubation with the secondary antibody. I used to have that problem with some proteins I was looking at and longer washing periods hugely improved the quality and signal-to-noise ratio on my images.
Hope it helps, take care!
I guess your antibody has some unspecific targets. Maybe the smear could be resolved if you run a big tris-tricine gel over night and even add some urea. Depends on what you are looking at there. To minimize unspecific interactions, maybe try a different blocking solution, decrease incubation times with the antibodies, or (if you don't already) have the antibodies incubate at room temperature.
I agree with arjan. it may well be a post-translational modification; poly-ubiquitination can easily cause smears, and this is even more often seen when proteins are overexpressed. cleavage is also not infrequent for overexpressed proteins and therefore the lowest band is most likely a degradation/cleavage product. dimerization looks less likely because the MW should be higher (although we do not know what the marker MW are). BTW, make sure you have enough protease inhibitors in your lysis buffer...
The above comments are, maybe, all correct but unless we run the MS-Spec otherwise we have no proof of anything. Yes, it is not uncommon to have multiple non-specific bindings using polyclonal antibodies. Sometime even transfection itself can generate sevearl unknown bands. Unfortunately, unless you have unlimited funding to identidy each one of them otherwise there is no way we can find out.
Smears in western blots sometimes are the result of too much product being loaded into the gel or In some other cases, when the antibodies are highly specific, do to the glycosylation state of the protein. If your protein of interest is highly glycosylated and you are just looking for expression levels and not post-translational modifications of the protein you can use de-glycosylation kits or compounds to clear up the smear.
As some of the comments above have stated, there is also the possibility that you might be looking at some protein degradation, or an antibody with some degree of non-specificity, in which case you may want to make sure that enough protease and phosphatase inhibitors are present in your buffer. You can also try washing longer with a high salt buffer right after incubation with the secondary antibody. I used to have that problem with some proteins I was looking at and longer washing periods hugely improved the quality and signal-to-noise ratio on my images.
Hope it helps, take care!
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