Dear Colleagues
I have recently run several 12% SDS-PAGE gels. I have loaded varying protein concentrations, ranging from 2.0 to 3.0mg/ml. I have stained the gels with Comassie blue. Would you expect to notice a major difference in band intensity between these concentrations?
Also, what procedure is the best for determining protein concentration in samples before loading onto the gel? I used the Nanodrop.
Thanks
If you use some image-quantifying software (e.g. Image J), you will probably be able to quantify a 1.5-fold difference in abundance. By eyeballing, I would rather trust a > 2-fold difference. As to using Nanodrop, it is OK if your protein is devoid of nucleic acid contamination: nucleic acids absorb ~10-fold more than proteins at 280 nm. But you can check for that by looking at the A260/A280 ratio: it should be about 0.5 for pure protein.
If you have loaded various amounts of protein per well, you can be able to distinguish them by naked eye, otherwise use an imaging software and perform some densitometry measuring.
Regarding the protein concentration, if I am not limited in sample, i avoid using Nanodrop as much as I can, since sometimes is not very reproductible.Most commonly, i perform a BCA assay to determine the concentrations of my samples.
Obviously, there will be different intensity protein bands on the gel. you can perform bradford test before loading on SDS-PAGE.
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