5X Bradford Method for Protein Concentration Measurement

The Bradford method is a fast, convenient, and sensitive method for protein concentration determination, widely used in biochemical and molecular biology research. This method relies on the principle that the Coomassie Brilliant Blue G-250 dye changes color when it binds to proteins under acidic conditions. The higher the protein concentration, the darker the color of the sample, and the protein concentration is estimated by measuring the absorbance. Here are the detailed steps for protein concentration determination using 5X Bradford reagents:

 

Sample Submission Requirements

1. Preparation of 5X Bradford Reagent

Purchase a ready-made 5X Bradford reagent or prepare it yourself according to the standard formula. If you prepare it yourself, ensure that the Coomassie Brilliant Blue G-250, methanol, acetic acid, and water are mixed in the correct proportions.

 

2. Preparation of Protein Standard Solution

Prepare a series of standard solutions using a protein of known concentration (such as bovine serum albumin, BSA) to draw a standard curve.

 

3. Sample Preparation

Dilute the protein sample to the expected testing range.

 

Analysis Workflow

1. Preparation of the Standard Curve

(1) Add a certain volume of the protein standard solution, such as 25 μL, in a 96-well plate or colorimetric dish.

(2) Dilute the 5X Bradford reagent to 1X, usually by mixing the 5X reagent with an appropriate amount of deionized water in a 4:1 ratio.

(3) Add an appropriate amount of 1X Bradford reagent to each well containing the standard solution, such as 200 μL, and mix well.

(4) Let it stand for 5-10 minutes to allow the color to fully develop, but not exceeding 60 minutes.

 

2. Sample Determination

(1) Add the protein sample to be tested in the same volume to another well or colorimetric dish.

(2) Add an appropriate amount of 1X Bradford reagent to each sample, same as the operation above.

(3) Mix well and let it stand to wait for color development.

 

3. Absorbance Measurement

(1) Measure the absorbance of each well or colorimetric dish at a wavelength of 595 nm using a spectrophotometer.

(2) The absorbance value of the blank control (containing only 1X Bradford reagent and water) should be subtracted from the readings of each sample and standard solution.

 

4. Calculation of Protein Concentration

(1) Draw a standard curve based on the absorbance values of the standard solutions, generally with protein concentration as the x-axis and absorbance as the y-axis.

(2) Use the absorbance value of the sample to find the corresponding protein concentration on the standard curve.

 

Precautions                                                                                                 

1. The bradford method may have different sensitivities to different proteins, which may affect the accuracy of the measurement results.

2. High concentrations of salt or other interfering substances may affect the measurement results, and it is recommended to dilute or pretreat the sample appropriately.

3. The color stability of the dye bound to the protein is limited, it is recommended to measure the absorbance as soon as possible after the color development is completed.

4. The bradford method is widely used due to its convenient operation, fast speed, and small sample size, and is one of the commonly used protein concentration determination methods in the laboratory.