BCA Assay Measures Protein Content

BCA (Bicinchoninic Acid, 2,2'-bicinchoninate) sodium salt is a light yellow powder that is hygroscopic and easily soluble in water or ethanol. BCA is a stable water-soluble compound that can bind highly specifically with Cu+ to form a purple complex. This complex has a maximum absorbance at 562nm and is proportional to the protein concentration.

 

Protein content refers to the total mass or quantity of protein in a sample, which can be determined by the BCA method. The principle is that under alkaline conditions, the protein can reduce Cu2+ to Cu+. The Cu+ forms a purple-blue complex with the BCA reagent (two BCA molecules chelate one Cu+). The intensity of the light absorption of this purple complex is proportional to the protein concentration, so the content of the protein can be calculated by measuring its absorbance at a specific wavelength.

 

Reagents Required

BCA reagent (store at room temperature), Cu reagent (store at room temperature), PBS (Phosphate Buffered Saline) (store at room temperature), BSA (Bovine Serum Albumin) standard - 5mg/mL (store at -20℃).

 

Detection Method

Microplate enzyme marker method.

 

Experimental Steps

1. Preparation of Working Solution

According to the number of standard and sample, prepare BCA working solution by adding 1 volume of Cu reagent to 50 volumes of BCA reagent (50:1), and mix well (there may be turbidity during mixing, but it will disappear after mixing). The BCA working solution is stable at room temperature within 24 hours (it is recommended to use it immediately after preparation).

 

2. Dilution of Standard

Dilute 10μL BSA standard with PBS to 100μL to achieve a final concentration of 0.5mg/mL. Add the standard in volumes of 0, 2, 4, 6, 8, 12, 16, 20μL to the protein standard holes in the 96-well plate, and add PBS to make up to 20μL in each hole.

 

3. Sample Dilution

Dilute the sample appropriately (it's better to do several gradients, such as 2-fold, 4-fold, 8-fold dilution), and add 20μL to the sample holes in the 96-well plate. (Due to the large error when the pipette takes a small amount of sample, the points in front of the standard line may not be accurate, so try to make the sample points fall after 1/2 of the standard line)

 

4. Content Determination

Add 200μL BCA working solution to each well, cover the 96-well plate with a plate cover, and place at 37°C for 15-30 minutes. Measure A562nm with an enzyme marker. Import the A562nm data of each dilution concentration standard into Excel, make a scatter plot, obtain the standard curve formula, and calculate the concentration of unknown protein according to the standard curve formula. (When incubating in a temperature box, be careful to prevent the detection results from being affected by water evaporation)