Horrendous background problem on chemiluminescent westerns

Frank O. Fackelmayer fof1 at chclu.chemie.uni-konstanz.de
Wed Sep 9 04:03:01 EST 1998


Try using your antibodies, both primary and secondary, at a much higher
dilution. Heavy background is mostly the result of too much antibody. As a
rule of thumb, use 1/10 the amount of primary antibody (in comparison to
colorimetric detection), and the lowest possible amount of secondary ab. In
our hands, the POX-coupled secondaries from sigma, diluted 1:100.000 (that´s
NOT a typo) in new blocking solution (TNT with 5% dry milk), works great. You
might have to titrate both antibodies, but it is really worth the work.

Frank



Guess wrote:
> 
> Hi.  I am hoping somebody can give some tips on cutting down the
> background on my chemiluminescent westerns (ECL reagent).  I get large
> spots, patches and smears all over (as opposed to a nonspecific
> recognition/banding pattern).  Its particularly bad  where the edges
> of the gel contact the membrane during transfer.
> 
> I have tried just about everything I can think of.  I wash extensively
> (sometimes overnite!) with TBS-T after the secondary incubation.
> Washes after the primary are for at least an hour with 4 changes of
> large quantities of TBS-T.  Both Ab incubations are done at rm temp.
> in fresh blocking sol'n.
> 
> I've tried blocking w/ 3% BSA, 5% NF dry milk, 1-3% gelatin,
> combinations of the above, and combinations of the above w/ various
> concentrations of PVP.  I have tried blocking at room temp, 37 deg, 4
> deg overnite and combinations of these for 30 minutes to hours to
> overnite.
> 
> We use Biorad pure Nitrocellulose and I've tried MSI nitrocellulose.
> The transfer is done in a tank w/ a standard buffer (Tris, glycine,
> SDS, methanol) for an hour to overnite at low voltage.
> 
> Anybody have some magical blocking solution??  ANY other insights
> would be appreciated too!
> 
> thanks!



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