I have a strange situation in hand and I wonder if anyone had a similar experience before....

you see, I have a HA-tagged protein that when expressed, show up as a 40kD protein.... and there is a faint band about ~43-45kD.... this faint band becomes a major band with MG132 addition to transfected cells.

So, it looks like ubiquitination....

But I cannot detect the ubiquitin (the upper band) using anti-Ubi antibody from Santa Cruz. Also tried anti-SUMO from Santa Cruz and it is notpositive as well.

any ideas, advice, or experience to share?

Thanks in advance!

My best guess is you a have glycosylation addition. either N-linked or O-linked. its hard to tell which type unless you know something about the addtion site or potential addition site.

Rb

R Bishop said:

My best guess is you a have glycosylation addition. either N-linked or O-linked. its hard to tell which type unless you know something about the addtion site or potential addition site. Rb

Thanks Bishop....

thing is this... I tried using the Pierce Kit for O-GlcNAC.... zilch. Nothing happening.

Running in silico analysis, there are potential O-glycosylation sites but no N-glycosylation sites...

So, still back to the drawing block....

Still open to ideas........

Cheers!

Ok,

See if you can borrow some PNGaseF and/or O-Glycanse from another lab to do a quick digest of your sample, much better than the kits.

Other than that I have no clue.

good luck

RB

R Bishop said:

Ok, See if you can borrow some PNGaseF and/or O-Glycanse from another lab to do a quick digest of your sample, much better than the kits. Other than that I have no clue. good luck RB

Thanks for the heads up!