Protein Conc using Bradford Assay - (Oct/25/2008 )

I am carrying out a transfection practical and I am using a Bradford Assay to determine the protein concenetration but I have never used a Bradford assay.

I assume I have to make a standard curve using BSA but what buffer do I use and what volumes? I believe we have to add 5ul of solution (standard BSA or test cell extract) to the Bradford assay solution but I don't now what concentrations of BSA to make up.

I hope that makes sense and any help would be really appreciated as I have never used this technique so I am working in the dark.

Don't be afraid, Bradford is not that complicated at all.

First, you use the same buffer for dilution of your standard protein (BSA), in which your sample that you will measure is in. So, lysis buffer or whatever it is. It has to be the same, so that the buffer itself won't interfere with your measurement and give the same background. I don't know which Bradford assay you use, but normally you have a 5x concentrated solution which you have to dilute with water 1:5 and then you add your protein, 5 µl is standard. So, you have 200 µl of Bradford + 795 µl water + 5 µl protein. The same without protein and just water to have a blank.

For your standard curve you should estimate in which range of concentration your amount of protein will be. Take 5 values which sorround your estimated value best. So maybe between 20 µg/ml and 2 mg/ml.

And you have to use disposable cuvettes, plastic ones. Be sure to mix the components well, as they will otherwise build up layering of colours. The darker blue your solution gets, the more protein you have. But don't put in too much, if your absorbance is above 1 (or below 0,1, measured at 595 nm), than your results are not reliable. In between this range is good.

Hope this helps for beginning.