Western Blotting using Anti α Tubulin antibody: Bands too high! - (May/25/2007 )

Hello everybody,

We have encountered some problems with Anti α Tubulin(H-300/SC:5546) in Western Blots. We are running 10% SDS-PAGE(Reducing) and after submarine blotting , we always see a band appearing at the hight of about 70kD,although it should be about 55kD!
The proteins were isolated from human and mouse heart sections and rat cell lines.
As secondary antibody , Rabbit anti-mouse IgG-HRP(DAKO P0161) is used and for detection, we use PIERCE ECL Western Blotting Substrate(#32106).
Is there somebody out there, who can explain this phenomenon?

Thanks for your help.
UMFARTOX

-UMFARTOX-

QUOTE (UMFARTOX @ May 25 2007, 02:55 PM)

Glycosylation of the protein maybe.

-aspergillie-

QUOTE (UMFARTOX @ May 25 2007, 01:55 PM)

size determination in SDS-gel is not precise, however, there is a strong difference between determined and expected size;

are the standard reference protein correctly assigned?

check a reference cell line for tubulin

I do not know much about PTM of tubulin but I suspect that this may not be of relevance in your case;

-The Bearer-

Plus, a 25 kDa PTM would be pretty huge

And I would ask : Is your blot clean? ie, does your antibody recognize many bands or just one?

-Madrius-