Or I should use elution buffer diluted 5X with dH2O and use it as blank OD. You are correct, after blanking the line should pass through 0. Meaning I have 2 blank for my assay. I substracted the OD with the blank before plotting the standard curve. So by theory any absorbance in the blank should have been zeroed. - posted in Protein and Proteomics: Hi all, Ive recently done bradford assay and am so confused if I did it right. Coomassie (Bradford) protein assay absorbance spectra. 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. Edited by Meg P. Anula, 13 October 2016 - 11:43 AM. It is fairly accurate and samples that are out of range can be retested within minutes. Bradford Protein Assay Introduction Use of the coomassie G-250 dye in a colorimetric reagent for the detection and quantitation of total protein was first described by Dr. Marion Bradford in 1976. Btw, I am also confused as in whether standard curve for Bradford should start from zero?   Best Answer. Let say I wanted to plot the standard curve in linear form. For standard, I used BSA serially diluted in dH2O, so obviously the blank is dH2O. What is the reason for not include zero in the straight line? It was in elution buffer containing imidazole, some salts and urea. 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. 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. bob1, Within the linear range of the assay (~5-25 mcg/mL), the more protein present, the more Coomassie binds. 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.... Edited by Meg P. Anula, 12 October 2016 - 10:39 PM. 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). You currently have javascript disabled. This is not recommended for shared computers, Sign in anonymously Please re-enable javascript to access full functionality. Thanks bob1. 23236). Bradford assay which is the blank? The Bradford is recommended for general use, especially for determining the protein content of cell fractions and assessing protein concentrations for gel electrophoresis. Don't add me to the active users list. The zero concentration will have zero OD after subtracting with blank (itself). ©1999-2013 Protocol Online, All rights reserved. I substracted the OD with the blank before plotting the standard curve. Remember me The Bradford assay relies on the binding of the dye Coomassie Brilliant Blue G250 to protein, in which the dye is proportional to the protein concentration. The blank and standards should be in the same buffer as your samples. I've recently done bradford assay and am so confused if I did it right. Scans of eight BSA standards (0 to 2000 µg/mL) tested with the Thermo Scientific Pierce Coomassie Plus Protein Assay Reagent (Part No. Best Answer But if the zero BSA (only buffer) is used as blank for all data. The thick line is the 2000 µg/mL sample. Since the blank is zero BSA concentration. It gives an easy way to estimate the protein concentration of a solution using a standard curve generated by the use of known concentrations of a protein. And I diluted the unknown protein sample with 5X dH2O. What you should have done is serially dilute the standard in your diluted elution buffer. Coomassie Plus (Bradford) Protein Assay Detergent Compatible Bradford Assay Coomassie (Bradford) Protein Assay; Features: Ready-to-use, reducing agent–compatible Bradford assay reagent that provides increased linearity of response and only half the protein-to-protein variation of other commercial Bradford assay formulations But what about the unknown protein? Bradford assay is a protein quantification protocol developed by Marion Mckinley Bradford in 1976. The method is based on the proportional binding of the dye Coomassie to proteins. 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. But what about the unknown protein? Several functions may not work. For standard, I used BSA serially diluted in dH2O, so obviously the blank is dH2O. The Bradford assay is very fast and uses about the same amount of protein as the Lowry assay.