Typically, seven concentrations of standards are prepared to cover the linear range of a Bradford protein assay, ranging from 0.125 to 2 mg/ml. The range of the standard curve for the Pierce Detergent Compatible Bradford assay is 4 times broader than the range for the Bio-Rad DC assay. With these principles of standard curve assays in mind, one can easily understand why it is neither necessary nor even helpful to know the actual amount (e.g., micrograms) of protein applied to each well or cuvette of the assay. If necessary, the accuracy of the standards can be increased by preparing the standard dilutions in the same buffer as the samples. 2.1 Standard Assay Protocol 11 2.2 Microassay Protocol 14 Section 3 Data Analysis 18 Section 4 FAQs and Troubleshooting 22 Section 5 Ordering Information 26 Section 6 References 28 Section 7 Appendix 30 4110065A.qxp 9/25/2007 2:39 PM Page 5. Any other standard may be chosen, but note that the same standard must be used in all experiments. Dilute the unknown samples in deionized water. Before using the standard curve you’ve generated you must be certain that the absorbance is a linear function of concentration, which holds within the limits of the Beer-Lambert Law.

In this experiment, the protein concentration of each dilution standard was determined by using the concentration formula as shown in Figure 4,where C1 is the concentration of Bovine serum albumin (BSA) standard (100 μg/mL), V1 is the volume of standard protein, C2 is the protein concentration determined in this equation and V2 is the total volume of the solution (1.5 mL). Like other protein assays, the result of the Bradford assay differs for different proteins. Comparing Pierce Detergent Compatible Bradford Assay to the Bio-Rad DC Protein Assay, better sensitivity is seen with the Pierce Detergent Compatible Bradford Assay using common detergents. Aim to 5-50 μg/ml. Standard Curve The Bradford assay is calibrated by using a pure protein of known concentration, called the "standard protein". Detailed Protocol for the Improved Bradford Protein Assay, using a Microplate Absorbance Reader: Prepare a 0.1 mg/ml stock solution of the standard, bovine serum albumin. You are aware that A 280 is about twice as sensitive to IgG as to BSA. Bradford (BioRad) Protein Assay 1.5 ml Protocol! 2 June 2015 Once you have performed the assay, a standard curve is generated and the results graphed.