1.2.2 BCA Assay

High Accuracy and Sensitivity, Good Reagent Stability, Detergent Compatibility, and Relatively Complex Operation

Experimental Procedures

1. Plot the Standard Curve

It is best to dilute BSA (Bovine Serum Albumin) with the same solution as the sample. Generally, for cost-effectiveness and convenience, distilled water can be used for dilution. Prepare the diluted BSA into standard solutions of different concentrations; it is recommended to set up more than 5 concentration gradients for the standards. The following concentration gradients can be used as a reference: 20, 125, 250, 500, 750, 1000, 1500 μg/mL BSA. Then add 20 μL of each standard solution to each well, prepare two sets of replicates for testing, and it is advisable to set 2–3 replicate wells for each concentration.
2. Preparation of BCA Working Solution
Mix Solution A and Solution B at a ratio of 50:1 to obtain the working solution. Note: When Solution B is added to Solution A, turbidity may be observed, which will disappear after gentle shaking; the working solution should be green.
3. Sample Detection
Add an appropriate volume of sample to a 96-well plate, and make up the sample volume to 20 μL with distilled water.
Add 180 μL of BCA working solution to each well (both standard curve wells and sample wells), and incubate at 37°C for 30 minutes.
4. Measure Absorbance at 562 nm and Plot the Standard Curve
Use a microplate reader to detect the absorbance of each well and record the data.
Generate the standard curve based on the absorbance values of the standard solutions, then calculate the corresponding concentration of the sample.