I want to detect the nuclear and cytoplasmic protein concentrations by using Bradford technique. I tried to many protocols, but the standards' values were not good enough (at 595 nm). I used BSA (at 7.5% concentration) as standard. Can anybody recommend an appropriate Bradford protocol that has been successful for you?
BSA is not a good standard protein for Bradford because of its low level of cystein. I suggest a mix of protein (eg. protein markers for PAGE). It works with our protein extract from plant tissues
Hi Bahadir,
What do you mean by not good enough? Are you only using the 7.5% concentration of BSA as standard? Usually a range of 5-7 different concentrations of BSA (you could choose values between 0.1% and 5%, for example) is used to construct a standard curve that allows you to determine in which range of concentrations the intensity of the color is proportional to the concentration (that is, the linear part of the curve). Remember you can always dilute your samples to bring them down to that range of concentrations.
As Daniel suggest you should use a range of concentrations of BSA to construct a standard curve. Depending on how much detergent you use in your cytoplasmic/nuclear extraction protocol, it might be better to switch to a BCA assay instead of Bradford, because the Bradford assay is sensitive to the presence of quite low concentrations of detergents. For Bradford the nr of Cysteine in your protein is not important (it binds mainly to Arg, Lys and His and isn't that redox sensitive) but for Lowry it can make a difference (BCA is sensitive to "free" Cys amino acids) . Pierce has a quite nice general overview about various protein assays:
http://www.piercenet.com/method/chemistry-protein-assays
Make a standard curve and plot it. You might balance for the detergents using a sample containing your lysis buffer (without extract) as a blank. BCA on microplates is the best choice in my opinion. Bradford has an advantage of fast measurement, if you have to check your protein in the middle of experiment, but is less accurate.
I believe for the bradford assay that it only works for certain protein compositions; the assay is extremely sensitive.
Try another protein assay if it is giving you trouble.
Dear Bahadir,
Are you using glass cuvettes for your assay. Sometimes such problems arise in Bradford if you are using glass cuvette. Eventhough 595 is a visible range you have to use quartz cuvettes. It will solve the problem. If you are using quartz cuvette, then the problem might be with your filtering. Good Luck.
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