Protein Quantification via Coomassie Brilliant Blue Assay

The Coomassie Brilliant Blue method for determining protein content is a commonly used method in biology and chemistry experiments. The basic principle is to use Coomassie Brilliant Blue under acidic conditions to stain the structure of proteins, and then determine the protein content in the sample through the ultraviolet spectrophotometric method.

 

In an acidic environment, hydrogen bonds and van der Waals forces exist between the complex of Coomassie Brilliant Blue and protein molecules, causing the color of Coomassie Brilliant Blue to turn from brown to blue. The protein content in the sample can be determined through the ultraviolet spectrophotometric method.

 

Experimental Steps

1. Sample Preparation

A suitable amount of sample is taken, and it is completely dissolved with protein dissolution solution to obtain the protein solution.

 

2. Addition of Coomassie Brilliant Blue

Add Coomassie Brilliant Blue solution to the protein solution and mix well.

 

3. Stand Still

Let the mixture stand for a while to allow Coomassie Brilliant Blue to fully bind with the protein.

 

4. Determine the Absorbance

Measure the absorbance value of the sample with a UV spectrophotometer.

 

5. Calculate the Protein Content

Convert the absorbance value to the protein content through the establishment of a standard curve.

 

Precautions

1. The solubility of the protein is very important in the experiment, so it is necessary to fully dissolve the protein when preparing the sample.

2. When adding Coomassie Brilliant Blue, it needs to be fully mixed to ensure that Coomassie Brilliant Blue is fully bound with the protein.

3. The determination of protein content requires the establishment of a standard curve, so a series of standard solutions of different concentrations need to be prepared before the experiment.

 

The above are the experimental steps and precautions for the Coomassie Brilliant Blue method for determining protein content. Through correct operations and accurate measurement, we can obtain accurate protein content results.