Description: The ATB BCA (bicinchoninic acid) protein assay kit provides a fast, easy and reliable way of protein concentration determination. This procedure is applicable to both microplate method and regular test tube method. The 500 ml kit is sufficient for 2500 microplate assays. The Principle of BCA protein assay is, under alkaline conditions, the peptide bonds in a protein can form complexes with Cu2+ ion. Followed by reduction of the Cu2+ to Cu1+, BCA reagent can react with Cu1+-protein complexes to produce a purple-blue product, which has strong light absorption at 562 nm. The BCA assay is used for the same reasons why the Lowry method is used. It has been suggested that the BCA assay will replace the Lowry because it requires a single step, in comparison with two steps in Lowry method.

Features:  Single step, in 45 min, 4 time faster than using Lowry method.

Highly sensitive, can detect protein concentration as low as 25 μg/ml, and protein 0.5 μg, when sample volume is 1-20 μl.

Can tolerate most detergent in sample, e.g. 5% SDS, 5% Triton X-100, 5% Tween20/60/80.

Show excellent linear relationship within the range of 20-2000 μg/ml.

Coefficient of variation for different protein is much lower than using Coomassie Brilliant Blue method.

Storage :  Solution A and Solution B store at RT. BSA Protein Standard ships at ambient temperature, for long term storage, store at -20°C.

PROTOCOL: Microplate Procedure

1. Prepare 4 - 6 serial dilutions of a BSA protein standard containing 200 μg/ml to about 2000 μg/ml protein. A standard curve should be prepared each time the assay is performed. For best results, the BSA standard should be diluted in the same buffer as the sample.

2. Prepare working solution by mixing 50 volumes of BCA Solution A with 1 volume of Solution B (50:1, Solution A:B. e.g. Mix 1 ml of Solution A with 20 μl of Solution B). BCA working solution is stable for at least 24 hours at room temperature.

3. Pipette 25 μl of each standard or unknown sample replicate into a microplate well.

4. Add 200 μl working solution to each well and mix well by gently pipette up and down several times. Alternatively, mix on a plate shaker for 30 sec.

5. Cover the plate, incubate at 37°C for 30-60 minutes. Alternatively, incubate at RT for 2 hours, or 60°C for 30 minutes.

6. Cool plate to room temperature. Measure the absorbance at 562 nm on a plate reader.

7. Calculate the sample concentration according to the standard curve.