strong 100kda bands for alpha tubulin + the usual 55 kda bands? URGENT - (Oct/01/2013 )

Hi,

Im working on GLUT-4 levels in baboon muscle, and I am using alpha-tubulin to normalise. I am getting two bands though, one strong band at about 100kda and the usual (less strong) 55kda that I am expecting. I am aware that alpha + beta tubulin is at roughly 100 kda. What possible reason could there be for two bands to consistently appear, even when the dilutions were checked and  rechecked? I dont think it is a blocking problem, as these two bands appear regardless of blocking bsa or milk for long periods. Any help would be much appreciated.

Thanks!

what were your dilutions based on?  Were they based on results from someone else working on baboon tissue?  If not, you may need to re-titrate the antibody concentration to get it working well for your system.  Could you do a peptide competition assay to determine if both bands are specific?

What exactly have you done? - I presume this is a denaturing gel, but what % gek? what's in your lysis and loading buffers?  How much protein did you load?  Are the antibodies new?  Commercial supplier?

I am using a 10% polyacylamide gel, using a RIPA buffer, and reducing sample buffer (2x) made up of upper tris, glycerol, sds, bromophenol blue and beta mercaptoethanol. I Ioaded 12 ul at 30 ug/ul concentration. The antibodies are mouse alpha tub from santa cruz that is relatively new. the samples are unboiled (as required for glut-4). thanks for your help!!

juliavv on Wed Oct 2 12:44:12 2013 said:

 the samples are unboiled (as required for glut-4). thanks for your help!!

This may well be the problem - could you try a less aggressive denaturation such as heating to 70 C for 10-20 minutes?