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strong 100kda bands for alpha tubulin + the usual 55 kda bands? URGENT


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#1 juliavv

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Posted 01 October 2013 - 12:00 PM

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!

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#2 bob1

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Posted 01 October 2013 - 02:56 PM

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?



#3 juliavv

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Posted 02 October 2013 - 04:44 AM

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!!



#4 bob1

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Posted 02 October 2013 - 11:33 AM

 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?






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