Hi.
Recently I began to extract protein from different rat organs: brain, lung, salivatory glands... using RIPA buffer and sonication. I use BCA for cuantification of protein, and I`m getting REALLY high protein concentrations, so, when I load them in gel, I have to use a dilution, however, I cannot see a band all the time. I`m looking for a 12kDa protein, so I use as a positive control an organ sample, since I cannot see the band all the time, is getting difficult to make sure is there. Does anybody have issues about this? I dilute the sample in destilated water for cuantification and western blot. Attached is 2 different western blots, in the first one I can see the protein (the three big bands) and in the second one, is not there, same sample, same dilution (1:10)
Hi Nancy --
First, the BCA assay gives an estimate of total protein concentration. You could have very high total concentration, but your protein of interest may still be lowly expressed. It may be necessary to do a smaller dilution, maybe 1:2 or 1:5, or none at all.
Second, 12 kD is a fairly small sized protein. Are you using nitrocellulose or PVDF as your membrane? You can order PVDF membranes that are optimized for smaller sized proteins.
Third, when you dilute your sample 1:10, do you add the water before or after the loading buffer is added? If you are adding the water after you might be diluting the loading buffer too much, affecting the glycerol levels and potentially leading to your sample floating out of the well.
Finally, what type of developing solution are you using? Is it an ECL reagent? Do you make it yourself in your lab or do you order it from a company? There are more highly sensitive reagents you might try.
Hi Samantha.
Yes, I`m using PVDF membrane from BioRad.
I add the water before the loaing buffer.
I`m using Pierce ECL.
Talking to my advisor she told me that maybe I`m not making a proper dilution for the BCA assay, since sometimes the absorbance is not in the linear range...
What concentration are your standards?
You can always dilute your sample (1:10) for the BCA assay. I usually have to do this because my protein concentration is too high and outside the 0.250 mg/ml - 2 mg/ml range of my standards.
Oh, also, we've had issues with the Pierce ECL solution not being sensitive enough. We now use the Pierce Pico PLUS substrate (or the Femto substrate for really low protein levels) which has given better results.
https://www.thermofisher.com/order/catalog/product/34580
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