Why the higher the concentration and lower the absorbance of protein measured by the Coomassie Brill

Does the protein in hyaluronic acid refer to the protein in a proteoglycan, and should you consider removing the glycosaminoglycans from it and purifying the protein, because Coomassie Brilliant Blue needs to be bound to the protein to develop color, and the glycosaminoglycans in the sample may have an effect, or the sample is contaminated.

Coomassie Brilliant Blue solution should be acidic and should not be used anymore if it turns blue-green.

In addition, tris, acetic acid, 2-mercaptoethanol, EDTA and other substances in the sample have a small amount of color interference on the experimental results, and the buffer can be used instead of distilled water when doing the standard curve, and the interference can be deducted, but a large number of detergents such as SDS have too much influence on the color and are not easy to remove, so we can only find a way to remove the detergent from the sample

Have you done a good job of the standard protein curve? I have done an experiment in protein determination in the past two weeks, the main reason for the irregular absorption peak of the determination should be the problem of the reagent, many proteins and brilliant blue on the market are of poor quality, and the effect of brilliant blue is not good even if it is imported, (the first reagent I bought, the absorbance of standard proteins cannot be measured) It is recommended that you find a larger company, change the reagent, the result should be good, the binding specificity of brilliant blue and protein is still very strong, this method should not be doubted.

The formula for calculating the content (%) is as follows: protein concentration (g ml) sampling volume (ml) absolute molecular line mass dilution factor (ml) 100%. Namely:

Content (%) Protein concentration ( g ml ) Sampling volume (ml) Absolute molecular mass Dilution factor (ml) 100% of which, the absolute molecular mass is the molecular weight of the protein, in general, the absolute molecular weight of 1 gram of protein is 1,000,000 micrograms, therefore, the absolute molecular mass of the protein is calculated as: protein mass (gram) 1,000,000 micrograms. The dilution factor is used to express the dilution factor of the sample, and in general, the dilution factor can be considered equal to the sample volume.

Therefore, the formula for calculating the protein content (%) is: protein concentration (g ml) sample volume (ml) protein mass (g) 1,000,000 micrograms sample volume (ml) 100%.

Coomassie Brilliant Blue Method.

The principle of determining protein concentration.

The Coomassie Brilliant Blue method uses protein dyes to determine protein concentration.

The principle of conjugation, a fast and sensitive method for quantitatively determining the concentration of trace proteins. This protein assay is being widely used because it has outstanding advantages over several other methods. This method is currently the most sensitive protein assay.

Coomassie G-250 dye binds to proteins in an acidic solution, so that the position of the maximum absorption peak (Lmax) of the dye changes from 465nm to 595nm, and the color of the solution also changes from brown-black to blue. The amount of protein bound to it can be determined by measuring the increase in light absorption at 595 nm. Studies have found that dyes are mainly associated with basic amino acids in proteins, especially arginine.

and aromatic amino acid residues.

The molecular structure is shown in the figure on the right.

Coomassie brilliant blue.

There are two types, G-250 and R-250.

Coomassie Brilliant Blue G-250 is red in the state of You Min Raiding Brother, and the maximum light absorption is 465nm; When it binds to a protein and turns cyan, the protein-pigment-binding bridge has maximum light absorption at a wavelength of 595 nm. Its light absorption value is directly proportional to the protein content.