Messy Western blots
Tue Sep 12 10:44:40 EST 1995
Sherilyn Bell <72650.1760 at CompuServe.COM> wrote:
>For some months I have been trying to examine the protein product
>of a DNA construct by transfecting cos-1 cells, lysing it in SDS
>sample buffer, running it on 8% acrylamide, blotting on
>nitrocellulose and incubating in anitbody against a portion of
>the construct. I have been detecting with ECL but my blots have
>many nonspecific bands. I have tried preclearing with
>untransfected cos-1 lysate, varying incubation times, washing for
>longer times and more changes (6x5min). I have been seeing the
>product I want, but its hard to interpret with so much
>background. The antibody is know to be specific against larger
>constructs containing the same sequence with less background.
>Does anyone have suggestions?
You should repeat the Western with secondary antibody alone to determine
whether the extra bands are due to binding of the primary or secondary
AB. If secondary you could preclear with non-conjugated Ig of the same
type together with protein A-sepharose beads.
Block the blots with 5% non fat dried milk in TBS-Tween and do all
washes and hybes in TBS-Tween. Titre out both antibodies, especially the
secondary AB. You can also dilute the ECL reagents if the required band
is coming up quite quickly and optimise the autorad exposure time.
Dept. of Biochemistry,
St Mary's Hospital Medical School,
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