Hi
I was wondering if anybody have used stain free gel (Biorad) to compare the expression of a protein of interest in Cytoplasm and Nucleus?
If I were to submit a paper including this stain free blot image to a decent journal, say Cancer Research, would that be any issue?
I am wondering whether I have to go for conventional normalization method(using loading control) for comparing proteins from different compartments in cells. While stain-free blot is easier to work with, I am worrying that reviewer would still like to see loading controls of nucleus and cytoplasm.
I think it's always a good idea to have loading controls. In your case, the question is not that you use a precast gel. You can always stain the gel to see if you have loaded equal amounts of all samples so that the difference you see in your western blot is not duo to loading of different amounts of samples. Also you want to see a good separation of cytoplasmic and nuclear proteins. So, it would be advisable to use standard loading controls that people published .
I agree with Hua. Without normalization of loaded proteins from the nuclear and cytoplasmic fractions, any observation is harder to interpret.
Hi Bumjim,
Thanks for your nice qeustion.
Just an addition to the previous answers, my advise is that always use positive control, in addition do stripping to the protein.
Best wishes
sarmad
Thanks everybody for a good suggestion.
Sarmad, I have a follow up question. I know it is always good to have a positive control if you have it. I was wondering, though, what is relation between using a positive control and stripping the membrane? Any further explanation on it please?
Hi Bumjun,
You are welcom.
The positive control, to prove your antibody is working. Whilst, the striping procedure is to prove the availabilties of your protien samples (if you get negative result).
Feel free for any further qeustion, please
Best wishes
Sarmad
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