I'm studying a GPI-anchored protein and all of the antibodies against this protein are ~5-7kD higher than the expected molecular weight (expected is ~18kD and most antibodies give ~25kD). I'm wondering if the reason for this may be due to the addition of the GPI anchor. I couldn't find out how much GPI anchors increase the molecular weight of a protein. Does anyone know if it's possible for them to increase the weight by ~5-7kD?
No the weight is based on a western blot. I wanted to do MS but can't until I'm sure the antibody is detecting the correct size. Thanks!
Jiahui is absolutely correct. There is quite a bit of literature on aberrant M.W. given by (especially) membrane proteins. If, however, your protein has no TM domains than other possibilities exist. For example, if your detecting your protein from native tissue/cell sources the M.W. shift could represent a splice variant or other post-translational modifications (glycosylation is common for secretory proteins). If your expressing the protein heterlogously the expression system could equally give you other PTMs. The bona fide way to confirm the protein sequence is mass spec - when in doubt go there. Also check does the M.W. of GPI anchors match the shift you're seeing?
Thank you for the responses everyone.
Stephen- The samples I'm using are cell culture lysates. This protein definitely does have a PTM (the GPI anchor), but I just wanted to see if there was any literature on how much that can increase the molecular weight. I couldn't find out anything about the MW of GPI anchors. I think I will do the mass spec as you suggested and compare the protein sequence.
Phil- I wanted to try to find this out first, because I have to make beads that have my protein of interest conjugated to them. Due to the mechanism of splicing and addition of the GPI anchor, the N and C termini of the protein get cleaved, and thus I can't use any tagged constructs (e.g. Myc), and corresponding beads.
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