Your Guide to Protein Quantitation

Protein quantification is divided into "total quantification method" of whole protein and
"individual quantification method" of specific protein according to its purpose. It is an
indispensable part of biological experiments. In order to verify the success of cell lysis, or to
compare or standardize multiple samples for parallel experiments, protein cytolysis should be
performed for protein quantification; in order to determine protein yield, the purified protein
should be quantified; In order to label the purified protein with biotin or reporter enzyme, the
protein sample should also be quantified to ensure that the labeling reaction is carried out at
the appropriate chemical concentration.

Quantitative analysis of proteins is an area of analysis often involved in biochemistry and other
life sciences. Accurate and reliable quantitative analysis of proteins in samples is a very
important task in biochemical experiments. Protein is a very important biological
macromolecule. It has many kinds of structures, heterogeneous structure, large molecular
weight and different functions. This brings a lot of concrete difficulties for the establishment of
an ideal and versatile method for quantitative protein analysis.

There are many methods for determining protein content.

1. According to physical properties: UV spectrophotometry

2. According to chemical properties: Kjeldahl method, biuret method, Folin-phenol reagent

method (Lowry method), BCA method, colloidal gold method.

3. According to the nature of dyeing: Coomassie blue staining, silver staining.

4. According to other properties: fluorescence method.

Protein quantitative analysis is also involved in many fields and industries of production
research, and is the most common method in biology, food testing and adulteration, clinical
testing, diagnosis of diseases and quality testing.

There are many methods for protein quantification, five of which are more common, namely
Bradford method, Bradford spot test, Coomassie spot test, ultraviolet spectrophotometry and
BCA method. Of course, the conditions for each method are different.

Bradford method
This method is quite accurate for most protein quantification, especially for small molecule
polypeptide quantification. Such as ribonuclease or lysozyme. However, a concentration of the
detergent exceeding 0.2% affects the quantitative results. Such as TritonX-100, SDS, NP-40
and so on.

Bradford spot test

This method is particularly useful for detecting elution components to localize protein eluates.
For example, the strength of the adsorption force of the eluate is detected.

Coomassie spot test

This method is particularly useful for detecting elution components to localize protein eluates.
For example, the strength of the adsorption force of the eluate is detected.

Ultraviolet spectrophotometry

Protein UV absorbance is the fastest of all protein quantification methods. Readings are
typically taken at 280 nm light wavelength. Its maximum absorbance at 280 nm light wavelength
is mainly determined by the presence of tyrosine and tryptophan. Absorption at the wavelength
of 205 nm light is also commonly used. Its maximum absorbance at 205 nm light wavelength is
mainly determined by the peptide chain, although amino acids also have an effect. In addition,
one major advantage is that the sample is not damaged when the protein concentration is
determined.

BCA method

Characteristics of BCA method: 1. High sensitivity, the lower limit of detection concentration
reaches 25μg/ml, the minimum detection protein amount reaches 0.5μg, and the sample
volume to be tested is 1-20μl. 2. Determination of protein concentration is not affected by
chemicals such as detergents in most samples, and can be compatible with up to 5% SDS in
samples, 5% Triton X-100, 5% Tween 20, 60, 80. 3. There is a good linear relationship in the
concentration range of 20-2000 μg/ml. 4. The coefficient of variation of different protein
molecules is much smaller than that of Coomassie Brilliant Blue. 5. Effect of chelating agent
and slightly higher concentration of reducing agent: EDTA is less than 10 mM. DTT is less than
1 mM, and mercaptoethanol below 1 mm.