Hello,
Can anyone, who is familiar with ADP-ribosylation, suggest me what is the best way to check if my protein is ADP-ribosylated?
Thanks.
Hi there,
If your protein is pure, MS will tell you if its MW is compatible with such modifications. If knowing the protein sequence, MS fingerprint might help you to identify the modification site.
Is it about single or Poly-ADP-Ribosylation (PAR)? As there are some commercial antibodies targeted to PAR, you might use WB...
Hi Dominique,
Actually it is one of my target proteins from proteomics which gives an additional higher band (around 20 kd higher and much more prominent and specific in IP) in western blot, this protein has several modification sites and I already ruled out the possibility for having phospho, glycosylation or acetyl PTM, the protein is known to be ADP-ribosylated (endogeneously), but I dont know what is the best way to confirm it/ to know what is the upper band.
Hi again with such shift in MW, it should be PAR therefore WB with appropriate antibody seems the easiest. An other thing to think of : if WB is positive for PAR then you might also consider action of PAR degrading proteins (like PARG) which are commercially available to show that degrading PAR is making your protein lighter in SDS-PAGE...
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