According to the literature there are three transcript variants at ~17, ~23 and ~26 kDa yet my most prominent bands from HepG2 whole cell lysates (suggested by manufacturer: Abcam for 26 kDa band) are at 50 and 55 kDa and one less prominent band at about 13 kDa (plus a full smear between the two if protein or Ab conc are increased).
I know there are also three N-linked-glycosylation sites on the protein, is all this variability simply due to varying levels of glycosylation?
And how can one be sure that the different bands between samples, at different MWs, are all CD63?
Although it is conceivable that an extensive glycosylation of a protein of predicted size of ~26 kDa could move on the gel slower to give a protein of size higher, it is unlikely. One of the way to make sure that the antibody is specific and can detect CD63 in immunoblotting specifically is to run a positive control and a negative control. The positive control could be an extract from cells that overexpress recombinant CD63 upon transfection of a plasmid that encodes for CD63. The negative control would be extract from untransfected or vector transfected cells. Once it is certain that the antibody is specific, one can determine whether other bands are specific or non-specific.
Hey there,
I currently have same problem and I talked to the company several times. So they suggested to me to use 'blocking peptide', provided from company as free, special to antibody of interest. Thus, you will able to know which bands is your interest.
I agree that if a blocking peptide is available for the antibody in question, it should allow determination about its specificity. However, it requires some optimization and an additional control peptide.
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