Larger than expected bands on a Western - What could they be? (Aug/04/2005 )
On my western I'm getting a band at the expected size but I'm also getting three larger bands. What could these larger bands be? I've done a competition assay using the peptide and the antibody and all the bands that are showing up on the western are specific. Also, I've tested the secondary antibody alone and the other bands are not due to non-specific binding of the secondary.
Whenever I've heard people try to come up with explanations for larger than expected bands they use a lot of hand-waving and mention post-translational modifications such as phosphorylation, glycosylation, etc. I'm not saying these aren't valid explanations but how do I go beyond the hand-waving and actually test if these larger bands are due to post-translational modifications?
Deglycosylate your protein and see what happens.
Whenever I've heard people try to come up with explanations for larger than expected bands they use a lot of hand-waving and mention post-translational modifications such as phosphorylation, glycosylation, etc. I'm not saying these aren't valid explanations but how do I go beyond the hand-waving and actually test if these larger bands are due to post-translational modifications?
Hydrolyse your purified protein by HCl and then perform HPLC for the putative carbohydrates, see for signal compared to your standard of putative carbohydrates.
what is the difference of mol wt. in these bands? Is it ubiquitination (you can Check with antibody)