Bradford assay which is the blank? - (Oct/05/2016 )

Hi all,

I've recently done bradford assay and am so confused if I did it right.

For standard, I used BSA serially diluted in dH2O, so obviously the blank is dH2O. I substracted the OD with the blank before plotting the standard curve.  

But what about the unknown protein? It was in elution buffer containing imidazole, some salts and urea. And I diluted the unknown protein sample with 5X dH2O. For my convenience, I used dH2O as my blank for unknown protein as well.

Is it correct?

Or I should use elution buffer diluted 5X with dH2O and use it as blank OD. Meaning I have 2 blank for my assay. One is dH2O for standard, another is diluted elution buffer for my protein.

What you should have done is serially dilute the standard in your diluted elution buffer. The blank and standards should be in the same buffer as your samples.

bob1 on Wed Oct 5 14:46:31 2016 said:

What you should have done is serially dilute the standard in your diluted elution buffer. The blank and standards should be in the same buffer as your samples.

Thanks bob1. Btw, I am also confused as in whether standard curve for Bradford should start from zero? Since the blank is zero BSA concentration

It depends... you can force it to go through 0,0 (that's 0 on X and 0 on Y) but to do so you need to apply a modification of the data, that should apply to all data points. Usually the equation will be something like Y= X³+X²+X + "a number"... this final number is the Y value where the line crosses 0 on the X axis (concentration =0). Many examples show the line going through 0,0, but in general there will be some absorbance for any sample including the blank at the measurement wavelength.

But if the zero BSA (only buffer) is used as blank for all data. The zero concentration will have zero OD after subtracting with blank (itself). So the line will definitely go through the origin (since x is 0 when y is 0). So by theory any absorbance in the blank should have been zeroed.

Let say I wanted to plot the standard curve in linear form. What is the reason for not include zero in the straight line? Is it more accurate? 

I did both method using the same data (pass thru zero and not pass thru zero), the protein concentration calculated was not to say very similar....

Sorry maybe I wasn't clear on my previous question. I understand (if we choose to include zero concentration on standard curve), then that's possiblity the trendline does not start from origin and gives the equation of y=mx+c instead of y=mx. But there are also some people think that standard curve should not include zero concentration as one of the data point. Is it wise to do so?

You are correct, after blanking the line should pass through 0. As to whether you should include 0 as a concentration in a standard curve is up to you, but I would look at where you expect your samples to lie relative to the standards. If they all lie close to 0 or are low concentrations then I would include 0 as a part of the curve, but would also include more low concentration standards and leave off high concentrations. You should also look at the guidelines for the kit and determine the effective concentration range - I doubt that 0 will be included in there.

Thanks so much @bob1.