I am running 38 mice samples via western blot for 2 targets. I have already finished data analysis and have found significant differences between treatment groups. If I normalized all proteins of interest to the total protein in each lane, is that sufficient?
I recently had a suggestion that said it was all wrong and I had to normalize to the control group in EACH BLOT. This would mean I would have to repeat all of these experiments.
Any suggestions?
Thanks in advance.
Hi Sarah,
Yes, you can run all the samples on one gel. Load up to 48 samples on a single E-PAGE 48 gel (ThermoFisher) mounted in a Invitrogen Mother E-Base Device.
Https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-gel-electrophoresis/protein-gels/specialized-protein-gels/epage-high-throughput-gel-system.html
The proteins can be transferred to membrane for Western blotting. It works well.
Best,
Mark Bernard
Hi sarah,
running control sample and corresponding treatment groups of same time points is advisable to compare results from same blot with target and loading control to ensure equal loading of total protein as you can avoid the variation among blots.
regards
RamaRao
I also would be very cautious to compare WB signals over different blots. Even when to gels are run in the same gel chamber and the same wet blotting tank and incubated with the same dilution of antibodies, I still often see differnces in WB signal which make quantitative comparision a problem. Blotting may be different for different sized proteins, so housekeeper staining won't help you.
So yes, you should have a control on each blot. Furthermore, I would be careful comparing signals from different blots even with normalization to a control group from each blot. Only trust what is on one blot. So either use big gels with a lot of wells, or first screen on different gels and then put the interesing samples on one gel together with the controls.
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