Hi everyone,
I am planning a series of Western Blot experiments where I will be probing for a range of targets in my samples. Whilst optimising, I have been cutting my membranes at the relevant molecular weights for each of my proteins to minimise amount of sample used.
However I know that some journals request images of the 'full' membrane in order to publish now and was wondering if an image of the membrane (cut into pieces) would be acceptable?
Has anyone had any recent experience with this/any advice to give?
If I can get away without running separate experiments for each protein, it would save me a lot of time (and money!).
Many thanks,
Rebecca
Read the journal guidelines (for those you intend to submit to) and ask the editors when in doubt. That's your safest bet.
While I understand the genuine reason behind the desire to cut the membrane, it is very questionable from a scientific perspective because you have decided the result before even doing the experiment. What about novel forms or related proteins? Opportunities for discovery could be missed. You could also miss a general non specific staining of the blot if you can’t see the whole m.w. range and get a false signal on a small piece of membrane. Making the whole blot available for review and then cutting the image electronically to save space in the journal would seem to be the best of both worlds. You don’t save any reagent admittedly.
Probe the whole membrane and use a "rainbow" western for several targets. Cutting the membrane does not save sample, as you need a certain amount in a band for detection. It also does not really save primary antibody, as that solution can be used many times (use 0.1% BSA in PBS as solvent with timerosal as preservative, develop blots at 4 degrees o/n). But you preserve the information about antibody specificity (that you get a single band).
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