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Western Blot Forum Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


Loading Controls for Membrane Fraction

Loading Controls for Membrane Fraction - Western Blot Forum

Loading Controls for Membrane Fraction - Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


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Old 01-30-2012, 03:05 PM
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Default Loading Controls for Membrane Fraction



Hi everyone,

I'm on the the hunt for a good loading control for blots of membrane fraction of proteins from mouse liver samples . Already tried e-cadherin and that really isn't working out (antibody works well but it looks like expression is influenced by our treatment, unless i've forgotten how to pipette entirely).

Any suggestions as to suitable targets/antibodies would be greatly appreciated.

Thanks
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Old 02-15-2012, 04:28 AM
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Default Re: Loading Controls for Membrane Fraction

Hi
A good method (the best) is looking at papers in the field (published already) using the technique and seeing what they used. This is very important as if you use one that is not widely acknowledged, the worse thing that can happen is you have to repeat all your experiments with the reviewers suggested loading control! not fun.

Some suggestions however include:

- having a cytosolic protein as as control for membrane purity
- a membrane protein lysate/recombinant purified protein as a control - the problem with a lysate or recombinant protein is you will need to strip and reprobe it as you know
-OR another method is using the recombinant protein with a stain on the gel / membrane.
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