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matt bruckert 09-18-2013 11:47 PM

Polyubiquitinated Protein
 
Hey guys 1st post. This place is great.

I have a bacterial protein (AnkB) that is 23kD. I think this protein is polyubiquitinated.
After transfection of flag-AnkB and treating the cells with MG132 (proteasomal inhibitor to prevent polyubiquitinated protein degradation) I purified AnkB with flag resin. I performed a WB on the IP elution. With the flag antibody I seen multiple bands all corresponding to AnkB + X ubiquitin moities (46kD, 54kD and 71kD). With the poly-UB antibody I had seen 1 band at 54kD (this same band was in the anti-flag WB)
However the literature always shows polyubiquitinated prtoteins as smears (does anyone know why its as smears?
I mean ubiquitin is 8.5kD so you should just have your protein + 8.5X for the polyubiquitnated forms. I wonder why the polyUB WB did not recognize the other bands the flag antibody did. Unless its not ubiquitinated, however since the 1 band showed up with poly-UB Ab I think it is.
TLDR. Why do polyuibiquitinated western blots have smears and the protein + the MW of the ubiquitin moeities attached.'
Thanks so much


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