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Western Blot - Blocking buffer

Western Blot - Blocking buffer - Molecular Biology Techniques

Western Blot - Blocking buffer - Molecular Biology Forum. Includes forums for common molecular biology techniques.


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Old 01-04-2011, 06:32 AM
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Default Western Blot - Blocking buffer



Hi,

I'm using 5% (w/v) bovine serum albumin to block my PVDF membrane after protein transfer. Thereafter I add my primary antibodies. The autoradiographs show non-specific binding. The target protein also shows but as a white blotch on the X-ray film. I tried reducing the antibody concentration and increasing the chemilumin reagents - with no success.

tshego
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Old 01-07-2011, 06:43 AM
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Default Re: Western Blot - Blocking buffer

Hola, I donīt know if your antibody is commercial or home made, in the first case the data sheet shows the adecuate dilution for WB, because your indications shows an excess of sample and primary abs, with low specificity too, as it wasnīt purified as a rabbit serum. In the same way the negatization of your main band shows an excess of ab and sample, I think. Bio-Rad has a blotter where the sample runs in a continous comb and after transference you put a chamber with different channels to use different ab concentrations to see the idoneuos, but if you or your institution havenīt it, you coudl apply the same sample (less than used in your WB with problems), cut different strips and incubate with different dilutions minor than used before, to find the ideal one. Buena suerte
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