I am trying to run a western probing for different molecules of the mTOR pathway. After BCA-ing and standardizing protein concentrations, I added the same amount of sample to my 6% and 10% gels but when I probe for housekeeping B-actin or a-tubulin, the 10% shows the expected same sized bands throughout the blot while the 6% has differential expression with each sample. I repeated the 6% and got identical results to the first time I ran it so I doubt its an artifact of some small mistake. Any ideas about what could be occuring?
Did you use a wet blotting equipment? I suggest to prepare the sandwich promptly after runing the gel, leave out any incubation in blotting buffer. Probably the lower range proteins leaked out the 6% gel. Try a higher mol weight loading control. Is your pre-stained ladder is OK after run?
Try different loading controls, gapdh or histone 3. I would expect different transfer conditions with a 6 and 10% gel as well, which can be optimized depending on the system being used. Are you using wet or semi-dry and what type of blotting paper. If you use NC paper then you can ponceu stain to check for loading by densitometry. Over-all there is a reason that you are using a 6% gel and we are not sure of the experiment, I would use the 10% gel unless you absolutely need the separation in lower Kd regions. If you need separation in the higher regions, I would suggest just run the gel longer and run out the lower Kd proteins.
hi Mona,
I wonder if you are using pre-cast gel (polymerized and ready to use) if so check gel expiration date. Once happened to me when I used expired gel. Or if you preparing your gel, before pouring mix well then let it polymerize otherwise lesser the concentration will likely to give you bigger transfer difference. And let us know, when you solve your trouble.
Goodluck!
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