Western trasfer and blotting troubleshooting

Dr Engelbert Buxbaum via methods%40net.bio.net (by engelbert_buxbaum from hotmail.com)
Mon May 4 13:24:10 EST 2009

Am 03.05.2009, 23:47 Uhr, schrieb shifali  chatrath  
<shifalich from rediffmail.com>:

> I transfered the protein on 12% Tris -Tricine gels to Methanol wetted  
> PVDF membrane using semi-dry apparatus at 300A, 9V for 40 min.
> Transfer buffer: 48mM Tris,Glycine 39mM pH(8.9) without adjusting,+ 20%  
> methanol.
> After transfer, markers on one side(first lane) got beautifully  
> tranfered but the other side of the gel (last lane),got diffused, can  
> anybody suggest why?Anyhow, I proceeded for blotting.

There is little point in proceeding with ab staining, if the transfer did  
not work. After wetting the PVDF with MeOH you need to transfer to  
blotting buffer, as MeOH is not conductive. The blotting sandwich needts  
to be assembled without air bubbles. Are the electrodes of the semi-dry  
blotter ok (not unevenly worn)?

> 3X washes in PBS. Then I added 2 Ab,anti-mouse preadsorbed human, 1:5000  
> in Blocking buffer, it got pptd. on ice and i notice only after adding  
> on blot.

Well, it shouldn't, so you must have done something wrong. The precipitate  
is probably not the antibody, as the concentration is too low to cause a  
visible precipitate. Redo the blocking buffer, making sure that you get  
the right chemicals in the correct amounts. If that doesn't get rid of the  
precipitation, leave the compounds out one by one to find the culprit. It  
could be some contaminant, or, in very rare cases, a bad batch of  
chemicals (Sigma once send me "SDS" that was water insoluble).

Alternative option: consider publishing in the "Journal of Irreproducible  
Science" ;-)

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