Experimental determination of protein concentration by bicinchoninic acid (BCA) assay

Summary

The bicinchoninic acid (BCA) assay is a new and improved Lowery assay that has recently been developed, with a simple reaction and virtually no influence of interfering substances. Source: Handbook of Protein Technology

Operation method

Diquillinic acid assay

Principle

In alkaline environment, the peptide bond structure of protein molecules can complex with Cu2+ to form complexes, and reduce Cu2+ to Cu+ at the same time. The BCA reagent can be sensitively and specifically combined with it to form a stable and colorful complex. It has a high light absorption value at 562nm. The color is directly proportional to the protein concentration, and the protein content can be calculated based on the magnitude of the absorption value.

Materials and Instruments

Protein samples to be measured
BCA NaOH Sodium carbonate Sodium bicarbonate Copper sulfate pentahydrate Sodium tartrate
Spectrophotometer 37°C Water bath Small beakers Pipettes Guns and spiking guns Test tubes Test tube racks

Move

1. Solvents

1.1 Reagent A, 1L

10 g BCA (1 %)

20 g _Na2CO3-H2O ) (2%)

1.6 g _Na2C4H4O6 (-2 _H2O ) (0.16%)

4 g NaOH (0.4%)

9.5 g _NaHCO3 (0.95)

Add water to 1L and adjust pH to 11.25 with NaOH or solid _NaHCO3.

1.2 Reagent B, 50 ml

2 g _CuSO4-5H2O (4%)

Add double-distilled water to 50 ml

Reagent A and Reagent B are stable for at least 12 months at room temperature and can be purchased commercially.

1.3 Standard Working Reagent (SWR)

50 copies of Reagent A

1 part of Reagent B

Reagent A and Reagent B are stable for one week.

2. Assay

2.1 Mix 1x volume of sample with 20x volume of SWR (e.g. 100 ul of sample to 2 ml of SWR);

2.2 Incubate at room temperature for 2 h (a) or at 37°C for 30 min (b);

2.3 Cool to room temperature in case (b);

2.4 Read optical density (OD) at 562 nm.

Caveat

1. To facilitate the reaction process of the BCA method, incubate the test tubes in a microwave oven for up to 20 s. 2. After the samples have cooled to room temperature, the test tubes should be incubated in a microwave oven.

2. After the sample is cooled to room temperature, the light absorption value increases by 2.3% every 10 min. After the sample has cooled to room temperature, the light absorption value increases by 2.3% every 10 min.

3. The BCA method has a sensitivity of 0.5-10 ug/ml for the detection of trace proteins, should be used as a concentrated reagent, and requires 60 min of reaction at 60 °C.

4. Compared with the Lowry method, the BCA method will significantly increase the light absorption value if the sample contains lipids.

5. BCA coloration changes in buffers containing sulfhydryl reagents and detergents.

6. most interfering substances are removed when protein samples are precipitated by DOC-TCA.

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