How to quantify animal tissue protein? An introduction to the Coomassie brilliant blue method

Why determine protein concentration first?

In animal experiments, many subsequent analyses need to be performed on the premise that the protein amounts are consistent. For example, to compare the expression level of a certain protein in the tissues of two groups of animals, if the total amount of protein added at the beginning is different, it is difficult to draw meaningful conclusions from the results. Therefore, after extracting protein from animal tissues or cells, it is usually necessary to first measure the concentration, and then adjust the amount used for each sample according to the concentration, so that the total protein amount in each reaction system is as close as possible.

Basic principle of the Coomassie brilliant blue method

The Coomassie brilliant blue method uses the color change characteristics of the dye after binding to protein. In an acidic environment, the Coomassie brilliant blue reagent itself appears reddish-brown, and after binding to protein it turns blue, and the depth of the blue color is positively correlated with protein concentration within a certain range. In experiments, a series of gradient solutions are usually prepared using standard protein of known concentration, the dye is added, the color signal is read, and a standard curve is drawn. The unknown sample is read under the same conditions and then compared with the standard curve to estimate the protein concentration.

Careful handling of animal samples is required

Animal tissue samples often have complex composition and may contain fat, blood, connective tissue, and so on. Before protein extraction, it is generally necessary to homogenize and lyse thoroughly, then centrifuge at low temperature, and take the supernatant for measurement. If more particles or lipids remain in the sample, the reading may be affected. Therefore, the choice of lysis buffer and the centrifugation step are relatively critical. A common practice is to use a buffer containing protease inhibitors to reduce protein degradation, while avoiding detergent concentrations that would severely interfere with the Coomassie brilliant blue reaction.

Common points to note during operation

The dye needs to be stored away from light, brought to room temperature before use, and mixed gently.

The standard curve and samples should preferably be measured on the same plate or in the same batch of reactions to reduce differences in conditions.

If the sample protein concentration is too high, it should be appropriately diluted so that the reading falls within the more reliable range of the standard curve.

Cuvettes or microplates should be kept clean to avoid fingerprints or residues affecting the results.

The Coomassie brilliant blue method is not the only method for determining protein concentration, but because it is simple to perform and relatively controllable in cost, it is relatively common in laboratories related to animal experiments. Mastering its basic principles and key operating points helps obtain more stable protein quantification results and lays the foundation for subsequent protein analysis of animal tissues.