Western blotting variability
jeffb at sccwrp.org
Sun Apr 6 10:56:39 EST 1997
I'm trying to get a semi-quantitative western blotting procedure to work using alkaline
phosphatase conjugated secondary antibody and image analysis. A 4 point standard calibration is
included in all gels, and the unknown samples are within the linear range. Three replicates of
each sample are run on three different gels.
The problem is with between blot variability (CV=25%). I have also tested within blot
variability, and this too can be high.
I'm using a BioRad mini-protean II electrophoresis setup (BioRad 10% ready gels)and mini
transblot module (overnight tank transblotting) with supported nitrocellulose.
Any suggestions to reduce the variability will be appreciated.
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