Reliable detection of differences with quantitative Western blots?
R.Jayakumar at RoswellPark.org
Wed Mar 17 10:59:56 EST 2004
Depends on the efficiency of transfer of proteins onto the blot, the percentage of gel (lesser percentage, better the uniformity of transfer), pore size of the membrane (smaller proteins are better retained by smaller pore size membrane), the voltage and the length of time, and months of optimization of conditions :-))). Also depends on the type of western aparatus you use. For general qualitative westerns, the procedures and errors are very forgiving, and wire electrodes like that used for Mini-Protean II (BioRad) will do, but such aparatus are plagued by lack of uniformity of electric field which will become evident once the bands are quantified and plotted, especially while using high methanol buffers and high percentage gels (>12%). Most people overcome this by using plate electrodes like the semi dry aparatus or the more popular Genei blotter. Anyway, I use the Mini-protean II aparatus with Immobilon P membrane (0.45 u) with a 15 % gel (not good gels for westerns, but I have to use it). At the best optimized conditions, I get upto 10 - 20 % variation between two bands of the same protein load loaded at different places on the gel. before optimization of conditions, I used to get 40 - 50% variation in densitometric analysis of bands. I use volume quantification of bands rather than Area quantification (densitometrically analyzed using a personal densitometer, molecular dynamics and quantified using Imagequant), since volume takes into account the intensity of the band as well. hence the reliable detection of differences may be well have to more than 20 % to be useful.
You might be able to improve this much further if you use plate electrodes which ensures more uniform electric field across the gel/blot sandwich and a low methanol buffer.
best of luck
> From: owner-methods at hgmp.mrc.ac.uk on behalf of Peter Frank
> Sent: March 17, 2004 10:21 AM
> To: methods at hgmp.mrc.ac.uk
> Subject: Reliable detection of differences with quantitative Western blots?
> What differences in protein expression can be reliably detected using
> densitometry on quantitative Western blots?
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