Commonly used protein detection methods in laboratories

For the most commonly used protein concentration measurement methods in laboratories , this article is enough! Choose the right method to double your experimental efficiency!

The tryptophan (Trp) tyrosine (Tyr) and phenylalanine (Phe) residues in proteins have UV absorption peaks near 280 nm by measuring the protein solution at 280 nm Based on the absorbance value at

Quick and easy

The operation is very simple and quick, just use a UV spectrophotometer to directly read the absorbance at 280nm

non-destructive

Samples can be recycled because they are not chemically modified or consumed during the measurement process

No reagents required

No additional chemicals required except buffer

Suitable for high concentrations of protein

Also relatively accurate in the high concentration range

Much interference

Nucleic acids (DNA, RNA) also have strong UV absorption at 260 nm, and also have certain absorption at 280 nm, which will interfere with the determination of proteins. Some other small molecule substances may also absorb at 280 nm, causing deviation in the results.

Lower sensitivity

For low-concentration protein solutions, the absorbance value may be too low and the error will be large.

Accuracy affected by protein composition

The tryptophan and tyrosine contents of different proteins vary greatly, resulting in large differences in their molar extinction coefficients. The average extinction coefficient is used for estimation. The accuracy will be affected by the amino acid composition of the protein itself. If the Trp and Tyr contents in the protein sample are abnormally high or low, the error will be greater.

Need pure sample

The sample cannot contain a large amount of substances that interfere with UV absorption, such as certain nucleic acid dyes, etc.

Coomassie Brilliant Blue G-250 dye binds to egg whites under acidic conditions, and the dye changes from the red form (maximum absorption wavelength at 465 nm) to the blue form (maximum absorption wavelength at 595 nm) nm) This color transition is caused by changes in the molecular structure of the dye after the dye binds to the protein. The binding is mainly through electrostatic interactions and van der Waals forces, especially the interaction between the sulfonic acid group of the dye and the non-polar region of the basic amino acid residues of the protein (such as arginine, lysine and histidine) and aromatic amino acid residues. The depth of the color (absorbance at 595 nm) is proportional to the protein concentration.

Higher sensitivity

Higher sensitivity than UV absorption method, suitable for measuring lower concentrations of protein

Easy and fast operation

The reagent is single, the operation steps are few, and the reaction is rapid, usually completed within a few minutes.

Less interference from salts and buffers in the sample

Compared with other colorimetric methods, there is less interference from common salt buffers in samples.

good stability

The dyed blue complex can be stable at room temperature for a period of time, making it easier to read data.

Standard curve dependence

It is necessary to use a standard protein of known concentration (usually bovine serum albumin from BSA) to prepare a standard curve in order to quantify the differences in the binding abilities of different proteins to dyes. Therefore, when using the BSA standard curve to measure other proteins, the results may be biased.

protein species dependence

Different proteins have different binding abilities to dyes, resulting in different quantitative results for different proteins. The measurement results of proteins with high content of basic amino acids and aromatic amino acids are relatively accurate, and vice versa, there may be larger deviations.

Reagents stain cuvettes

Coomassie Brilliant Blue dye tends to stain glass or plastic cuvettes, so you need to handle it with caution or use disposable cuvettes.

Limited linear range

At higher protein concentrations, the reaction may saturate, resulting in a nonlinear standard curve.

Sensitive to detergents

Some detergents, such as SDS, can interfere with the Bradford reaction

The Lowry method is a classic protein quantification method based on two consecutive chemical reactions. The first step is that the peptide bonds in the protein react with copper ions under alkaline conditions to form a complex, and the copper ions are reduced to cuprous ions. The second step is that the reduced cuprous ions reduce the FolinCiocalteu reagent (a mixture of phosphomolybdic acid and phosphotungstic acid), causing the reagent to develop color and produce a blue color. The depth of the color (absorbance is usually measured at 750nm or 660nm) is proportional to the protein concentration.

Higher sensitivity

More sensitive than UV absorption method and Bradford method, can detect lower concentration of protein

Relatively little affected by protein composition

Because it is based on peptide bond reaction, it is less affected by the amino acid composition of different proteins than the Bradford method.

The operation steps are complicated

Requires multiple reagents, many steps, and takes a long time

Reagents are unstable

Folin-Ciocalteu reagent is unstable and easily contaminated by reducing substances and needs to be prepared before use.

Much interference

Susceptible to interference from a variety of chemicals, including some buffer components (such as Tris, EDTA), and non-ionic detergents and reducing agents. The purity of samples and reagents needs to be strictly controlled.

time dependence

Color development reactions require precise control of reaction time and temperature. Reaction times that are too long or too short will affect the accuracy of the results.

Standard curve dependence

It is also necessary to use standard proteins to prepare a standard curve

The principle of the BCA method is similar to the Lowry method. It is also based on a two-step reaction. The first step is that the peptide bonds in the protein reduce copper ions to cuprous ions under alkaline conditions. The second step is that the cuprous ions react with the BCA reagent (bicinchoninic acid) to form a purple BCA-Cu* complex. This complex has a maximum absorption at 562 nm. The depth of the color is proportional to the protein concentration.

High sensitivity

The sensitivity is equivalent to the Lowry method and higher than the Bradford method and UV absorption method.

Relatively easy to operate

The reagents are relatively stable, usually provided in premixed form, and the operating steps are simpler than the Lowry method.

Less interference from detergents

Compared with the Lowry method, it is less interfered by some detergents (such as SDS, Triton X-100) and is more suitable for samples containing detergents.

Wider linear range

The linear range of the BCA method is wider than that of the Bradford method

good stability

The developed complex is more stable than the Bradford method and can be left for a longer period of time before reading the data.

Standard curve dependence

Need to use standard proteins to prepare a standard curve

Copper ion reduction reaction

Based on copper ion reduction reaction, easily interfered by reducing substances

time and temperature dependence

The reaction requires heating and incubation, and the results are affected by time and temperature.

Sensitive to lipids

Lipids can interfere with BCA reactions

The biuret method is one of the earliest methods used for protein quantification. Under strong alkaline conditions, compounds containing two or more peptide bonds (including proteins) can react with copper ions to form a purple-red complex. The maximum absorption wavelength of this complex is around 540nm. The depth of the color is proportional to the protein concentration.

Minimally affected by protein composition

Because it reacts directly with peptide bonds, it is minimally affected by differences in the amino acid composition of different proteins, and the quantification of different proteins is relatively accurate.

Less interference

Less interference from common salt buffers and some small molecular substances in samples

Low sensitivity

Compared with the Bradford method, the Lowry method and the BCA method, it has the lowest sensitivity and can usually only be used to measure higher concentrations of protein (>1 mg/mL).

Need a lot of samples

Due to low sensitivity, higher concentrations of protein samples and larger amounts of sample are required to obtain readable absorbance values.

More steps

Reagents need to be prepared and the steps are relatively cumbersome.

Standard curve dependence

Need to use standard proteins to prepare a standard curve

In all aspects of laboratory protein detection, the quality of experimental consumables has an important impact on the reliability of test results. Aijin Biotech is rooted in the field of life sciences. With years of accumulated technology and continuous innovation, it has a series of consumables adapted to protein detection, including ordinary suction tips (low adsorption models) enzyme plate microcentrifuge tubes, conventional centrifuge tubes, cell culture dishes, culture plates, culture bottles, etc.