BCA Protein Assay Kit

Colorimetric BCA protein assay for microplates: 37°C for 30 min, read at 562 nm, BSA standard curve 0–300 μg/mL (R² ≥ 0.99).

Product overview

BCA Protein Assay Kit is designed for accurate determination of protein concentration in test samples. In an alkaline environment, proteins reduce Cu²⁺ to Cu⁺, and 2,2′-bicinchoninic acid (BCA) specifically chelates Cu⁺ to form a stable purple complex. Within an appropriate concentration range, color intensity is directly proportional to protein concentration. By generating a standard curve with the protein standard supplied in the kit and using a colorimetric assay, protein concentration in test samples can be determined rapidly and accurately.

Assay at a glance

Detection	OD₅₆₂, microplate reader set to 562 nm
BCA working reagent	Reagent A and Reagent B mixed at a volume ratio of 50:1 immediately before use
Per well	50 μL standard or diluted sample + 200 μL BCA working reagent, in duplicate
Incubation	37°C for 30 min, then cool at room temperature for 10 min
Standard curve	BSA, 0–300 μg/mL (8 standards diluted from the 1 mg/mL BSA Standard)
Performance	R² ≥ 0.99; RSD of duplicate measurements ≤ 10%
Storage	BSA Standard at -20±5°C; Reagent A and Reagent B at 5-30°C

Protein samples lysed with RIPA lysis buffer (Strong, Medium, Weak, or the RIPA Lysis Buffer Set) can be quantified with this kit.

Features and applications

Features

This kit provides stable and reliable performance. The linear fitting coefficient of the standard curve is R² ≥ 0.99, the relative standard deviation (RSD) of duplicate measurements for standards and test samples is ≤ 10%, and the in-house quality-control sample error is ≤ 10%, helping ensure accurate and dependable protein quantification.
Designed for microplate reader detection. If suitable accessories are available in the laboratory, a UV-visible spectrophotometer may also be used for absorbance measurement.

Applications

Protein concentration determination in test samples.
Quantification of cell and tissue lysates prepared with RIPA lysis buffer before Western Blot, immunoprecipitation (IP) and ELISA.
Sample normalization before electrophoresis or Western Blot.

Protocol

Notes

For Research Use Only. Not for use in diagnostic procedures.
Mix thoroughly before use. Avoid repeated freeze-thaw cycles.
In the BCA assay, color development continues to increase over time and the reaction rate is temperature dependent. During sample measurement, keep the laboratory temperature as constant as possible and strictly control the reaction and measurement times.
This manual is designed for microplate reader detection. If suitable accessories are available in the laboratory, a UV-visible spectrophotometer may also be used for absorbance measurement.
Avoid repeated freeze-thaw cycles of the protein standard. Use it as soon as possible after opening.
For your safety and health, please wear a lab coat and disposable gloves when operating.

Assay procedure

Preparation of BCA working reagent: according to the number of samples to be tested, mix BCA Reagent A and Reagent B at a volume ratio of 50:1 immediately before use.
BSA protein standards: prepare a microplate and dilute the 1 mg/mL BSA standard according to the table below. Add the standards to the wells in duplicate.
Vial 1 mg/mL BSA (μL) ddH₂O (μL) Final Protein Concentration (μg/mL)
STD 1 0 50 0
STD 2 1 49 20
STD 3 2 48 40
STD 4 4 46 80
STD 5 6 44 120
STD 6 8 42 160
STD 7 10 40 200
STD 8 15 35 300
Dilute the test sample appropriately so that the protein concentration of the diluted sample falls within the linear range of the standard curve. Add 50 μL of each diluted sample to the microplate in duplicate.
Add 200 μL of BCA working reagent to each BSA standard well and each test-sample well, then shake to mix thoroughly.
Incubate the microplate at 37°C for 30 min.
Remove the microplate and cool it at room temperature for 10 min.
Measure the OD₅₆₂ value of each sample with a microplate reader set to 562 nm.
Data processing:
1. Plot the OD values of the BSA protein standards on the y-axis against the corresponding protein concentrations on the x-axis to obtain the standard linear equation.
2. Substitute the OD value of each test sample into the standard equation to calculate the initial protein concentration, then multiply by the dilution factor to obtain the final protein concentration of the test sample.

Vial	1 mg/mL BSA (μL)	ddH₂O (μL)	Final Protein Concentration (μg/mL)
STD 1	0	50	0
STD 2	1	49	20
STD 3	2	48	40
STD 4	4	46	80
STD 5	6	44	120
STD 6	8	42	160
STD 7	10	40	200
STD 8	15	35	300

Components

Components	BR4C201-01 250 T	BR4C201-02 500 T	BR4C201-03 2500 T
BSA Standard (1 mg/mL)	2×1 mL	4×1 mL	20×1 mL
Reagent A	50 mL	100 mL	5×100 mL
Reagent B	1 mL	2×1 mL	10×1 mL

Shipping and storage

Condition	Recommendation
Storage	Store BSA Standard (1 mg/mL) at -20±5°C. Store Reagent A and Reagent B at 5-30°C. After transport at 2-8°C, store each component under its specified storage condition upon receipt.
Shipping	Ship under conditions that maintain each component's specified storage condition.
Handling	For research use only. Review the MSDS before use.