Western blotting variability

Jeff Brown 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.
Thanks you

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