Hi, in the Bradford assay you have to prepare a standard curve, in particular you have a linear range of the assay for your standard protein, for example BSA, between 1 to 20ug or 20-140 ug. In the standard curve you have to add the ul of lysis buffer in which you resuspended your protein of interest. The same ul of buffer the you add to your blank sample. In your situation, if I understand correctly, the buffers are different (prepared with completely different reagents), so you have to prepare a blank and curve samples for each buffers, in this case 3 curves and 3 blanks (all in duplicates at least). Another way, not properly correct, it would be to prepare only a standard curve, without adding any buffers, only water, and prepare at least 3 different blanks.

Hi, in the Bradford assay you have to prepare a standard curve, in particular you have a linear range of the assay for your standard protein, for example BSA, between 1 to 20ug or 20-140 ug. In the standard curve you have to add the ul of lysis buffer in which you resuspended your protein of interest. The same ul of buffer the you add to your blank sample. In your situation, if I understand correctly, the buffers are different (prepared with completely different reagents), so you have to prepare a blank and curve samples for each buffers, in this case 3 curves and 3 blanks (all in duplicates at least). Another way, not properly correct, it would be to prepare only a standard curve, without adding any buffers, only water, and prepare at least 3 different blanks.

Thanks, Valeria,

At the first times, I use mQ to blank and dilute all samples. After that, I think I should be chosen other buffers for every sample. With elution sample, I know that elution buffer is the best choice. But with the fermentation sample, I really confused. The BSM medium has no color, but after fermentation procedure, it becomes to be yellow. Therefore, it's hard to decide which buffer for fermentation and flow-through sample.

Hi, I understand the problem. If you perform an analysis before and after the fermentation, at least you can compare the results or see if you have different absorbance between the blank. You have to known if the buffer after fermentation interfere with the assay. For the flow-through sample I am not sure if you consider the sample before the eluition step, so what kind of sample/buffer do you have, it is yellow? In this case as before if you simple make a reaction with Bradford how canhe the color of the assay and the blank absorbance?

For your work it is necessary the results for all the three experimental points, or you need to compare the results as an internal experiment control?

Why do you need to know (total) protein concentration in the "fermentation" broth? You cant say if its only your protein of interest in many others... If you try to calculate efficiency run an SDS-PAGE of the different stages (if needed after precipitation). Even better, if you have a specific antibody, do a western blot.

Yoram

Hello! generally, the different buffers should not interfere with the assay, but if you would like to check that, prepare the standard curves with each buffer. Take into account that the sample volume added to the Bradford reagent should not be higher than (aprox.) 2% of the reagent volume. There are certain interfering compounds that you should check its presence and concentration (e.g. SDS, Triton). Also, if the concentration of eluted protein (collected from the column) is too low, you might not see the "peak", in which case you should change the method (maybe UV absorption). Good luck!

Maybe my lab protocol will help you.

Hi, Please find the attached file, may be help you 

Thanks, Valeria. With your advice, I think I have to have many standard curves for many different samples. Now, the best choice of buffer to blank for broth and flow-through sample is mQ, I think.

Thanks, Facundo. In my current protocol, 300 ul sample will be added to 1200 ul reagent. Do you think it is a problem when the maximum sample of your that is lower 2% of Bradford solution? And I'm afraid that the elution sample contains high salt concentration that interferes the assay.

Thanks, Yoram. I want to know the total protein concentration to figure out the performance of purification process by using the amount of desired protein after and before purification. SDS-PAGE I just know the purity of samples, and percent of each protein in the lane. And Western blot just confirms the present of my interested protein.

Thanks, Krzysztof. Thanks, Mohamed. Which membrane do you often filter your Bradford reagent? I see many protocols advise to use Whatman filter paper. Unfortunately, I have run out all these papers, so I think of using Minisart filter 0.45 um (Cellulose acetate membrane). Some people told me that Coomassie would bind onto cellulose acetate, so if I use this filter, the color of reagent will be lost. I don't know. And one question, why do you use PBS for dilution 

Hello! If you dilute too much the Bradford reagent, the efficiency of coomasie colorant to bind to the protein will be poor, because your are changing the reaction medium. You could correct this problem using a concentrated Bradford reagent, taking into account the final volume of your mix. If you think that your (high concentrated) salts in your elution buffer could interfere, you could check that with the calibration curves. If Bradford reagent still remains being a trouble to you, I recommend you to use UV quantification. Goof luck!

I usually use PBS, because we usually do have it at hand. As I wrote, other buffers can be used, especially if you know your protein preferences for pH, salt concentration, specific ions requirements, but that is important mostly for purified protein preparations, not for total proteins. If you run out of whatman filter paper, use coffee filters, just wash them in water several times and let them dry before using.

use unknown buffers or distilled water