Horrendous background problem on chemiluminescent westerns

Guess ckafer at iastate.edu
Tue Sep 8 14:29:22 EST 1998


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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