Micro BCA Protein Assay Kit

List of Components

Description	Quantity	Volume	Contents	Catalog Number
Micro BCA Reagent A	1	100mL	/	AR1110-A
Micro BCA Reagent B	1	100mL	/	AR1110-B
Micro BCA Reagent C	1	5mL	/	AR1110-C
Albumin (BSA) Standards	1	20mL(2 mg/ml)	containing bovine serum albumin (BSA) at 2mg/mL in 0.9% saline and 0.05% sodium azide	AR1110-D

Overview

Form Supplied	Micro BCA Reagent A, B, C: ready-to-use 1X solutions BSA Standard: stock solution for preparing a series of standard protein dilutions
Assays per kit	Tube procedure: 200 assays Microplate procedure: 1300 assays
Storage	Upon receipt store at room temperature. It is stable at room temperature for one year.
Note	If either Reagent MA or Reagent MB precipitates upon shipping in cold weather or during longterm storage, dissolve precipitates by gently warming and stirring solutions. Discard any reagent that shows discoloration or evidence of microbial contamination.
Assay Range	Test Tube Procedure (linear working range of 0.5-20µg/mL) Microplate Procedure (linear working range of 2-40µg/mL)
Equivalent	Thermofisher (Product No. 23235), Test Tube Procedure (linear working range of 0.5-20µg/mL) Microplate Procedure (linear working range of 2-40µg/mL) Millipore Sigma (Product No. QPBCA), Test Tube Procedure (linear working range of 0.5-30µg/mL) Microplate Procedure (linear working range of 1-20µg/mL)
Compatibility with reagents	Compatible with typical concentrations of most ionic and nonionic detergents Incompatible with chelating agents, strong acids or bases, and reducing agents (e.g. copper reductants): interfere with the reduction and chelating reactions of the assay mechanism
Applications	Western blotting, protein expression assays, protein profiling and characterization, protein quantitation assays *Our Boster Guarantee covers the use of this product in the above tested applications.
Physical State of reagents mix	Light blue liquid
Reagent Color/Absorbance	Blue / A562 nm
Activity	Active in alkaline medium
Description	Micro BCA Protein Assay Kit is a ready-to-use, detergent-compatible, Western blot related, total protein analysis reagent, used for the quick determination of total protein concentration of dilute protein solutions by measuring absorbance at 562 nm and comparing to a protein standard absorbance vs. concentration curve. The Micro BCA Protein Assay Kit provides an ultra-sensitive method to detect and quantify protein concentration. The kit has been optimized for determining total protein content in low protein concentration samples (0.5-20 μg/ml). The kit is sensitive to copper reductant and chelator instead of to both ionic and non-ionic detergents.
Cite This Product	Micro BCA Protein Assay Kit (Boster Biological Technology, Pleasanton CA, USA, Catalog # AR1110)

Assay Information

Assay Type	Protein Quantitative Measurement
Sample Type	Serum, Plasma, Cell culture extracts, Tissue Extracts
Technique	Solution-based detection of spectral absorbance at 562 nm; Smith Protein assay
Detection Method	Colorimetric/Spectroscopic
Equipment needed	Test tubes or miscroplates; Spectrophotometer; Microplate Reader
Assay Purpose	Measure protein concentration in solution
Incubation conditions	60 min at 60°C

Assay Principle

Micro BCA Protein Assay Kit is a ready-to-use, detergent-compatible, Western blot related, total protein analysis reagent, used for the quick determination of total protein concentration of dilute protein solutions by measuring absorbance at 562 nm and comparing to a protein standard absorbance vs. concentration curve.
The Micro BCA Protein Assay Kit provides an ultra-sensitive method to detect and quantify protein concentration. The kit has been optimized for determining total protein content in low protein concentration samples (0.5-20 μg/ml). The kit is sensitive to copper reductant and chelator instead of to both ionic and non-ionic detergents.

Usage

Studying protein-protein interactions, Measuring column fractions after affinity chromatography, Assessing protein yields in whole cell lysates, High-throughput screening of fusion proteins.
To be used with: Lysates or homogenates, prepared with non-reducing lysis and extraction buffers: Mammal Tissue Protein Extraction Reagent, Mammal Cell Protein Extraction Reagent, Mitochondria Extraction Reagent, Membrane Protein Extraction Kit, Cytoplasmic and Nuclear Extraction Kit, Bacterial Cell Protein Extraction Reagent
For RIPA lysis buffer, dilution of 1:10 with distilled water is necessary to reduce the interference. It is recommended that same diluent should be used to make the BSA Standard solutions as that of the protein samples.

Background

The BCA Protein Assay is a detergent-compatible formulation based on bicinchoninic acid (BCA) for the colorimetric detection and quantization of total protein. This method combines the reduction of Cu 2+ to Cu 1+ by protein in an alkaline medium (Biuret reaction) with the highly sensitive and selective colorimetric detection of the cuprous cation (Cu 1+) by bicinchoninic acid, forming a purple-colored BCA/copper complex with absorbance maximum at 562 nm. Measurement of A562 is used to determine the concentration of protein in solutions. Protein concentrations are estimated by reference to absorbances obtained for a series of standard protein dilutions (e.g. of BSA), which are assayed alongside the unknown sample. The Micro BCA Assay uses concentrated solutions and extended incubation times for the detection of dilute protein samples.
The BCA assay involves two consecutive steps of color development, where the final product indirectly depends on the protein concentration in the solution. The first step is the chelation of copper with protein in an alkaline environment, followed by reduction of the Cu2+ (cupric) to Cu1+ (cuprous) cations forming a light blue complex. It has been shown that cysteine, cystine, tryptophan, tyrosine, and the peptide bond are able to reduce Cu2+ to Cu1+. The amount of reduction is proportional to the protein present. In the second step of the color development reaction, bicinchoninic acid (BCA), a highly specific chromogenic reagent for Cu1+, reacts with the reduced cuprous cation that was formed in step one, producing a purple complex with strong absorbance at 562 nm (Smith 1985, Wiechelman 1988). The purple-colored reaction product results from the chelation of two molecules of BCA with one cuprous ion. The intensity of the BCA/copper purple complex is proportional to the amount of protein in the sample. Thus measuring the intensity of the absorbance at 562 nm is proportional to protein concentration. The BCA protein assay demonstrates higher tolerances towards common interfering substances, such as nonionic detergents and buffer salts, than the Lowry technique.

Assay Protocol

Preparation of Standards and Working Reagent (required for both assay procedures)

A. Preparation of Albumin (BSA) Standards

Follow Table 1 to prepare a fresh set of standards. It is recommended that same diluent should be used to make the BSA Standard solutions as that of the protein samples.

Table1. Preparation of Diluted Albumin (BSA) Standards

Tube Number	Volume of Diluent (μl)	Volume of BSA (μl)	Final BSA Concentration (μg/ml)
A	980 μl	20 μl of 2 mg/ml Stock	40 μg/ml
B	400 μl	400 μl of tube A	20 μg/ml
C	400 μl	400 μl of tube B	10 μg/ml
D	400 μl	400 μl of tube C	5 μg/ml
E	400 μl	400 μl of tube D	2.5 μg/ml
F	600 μl	400 μl of tube E	1 μg/ml
G	400 μl	400 μl of tube F	0.5 μg/ml
H	800 μl	0	0 μg/ml =Blank

B. Preparation of BCA Working Reagent

1. Use the following formula to calculate the total volume of working reagent required: (well numbers for standard + well numbers for sample) × (times of repetition) × (volume of working reagent per well) = total volume of working reagent required

For example: For the standard Test Tube Procedure, each sample needs 3 dilutions and 2 replicates:

(8 standard wells + 3 sample wells) × (2 replicates) × (1 ml) = 22 ml working reagent required (round up to 25 ml)

Note: Each sample replicate requires 150 μl of BCA working reagent for each well in a microplate assay or 1 ml for a test tube procedure.

2. Mix Micro BCA Reagent A, Micro BCA Reagent B and Micro BCA Reagent C in the ratio of 25:24:1. i.e., mix 5 ml of Micro BCA Reagent A and 4.8 ml Micro BCA Reagent B with 0.2ml of Micro BCA Reagent C.

Note: When Micro BCA Reagent C is initially added into Micro BCA Reagent A and Micro BCA Reagent B, turbidity occurs that quickly disappears upon mixing to yield a clear-green solution.

Assay Procedure

• Test Tube Procedure

1. Add 1.0 ml of each standard and protein samples into separate labeled test tubes.

2. Add 1.0 ml of BCA working reagent to each tube and mix well.

3. Seal tubes and incubate at 60°C in a water bath for 1 hour.

4. Cool all tubes to room temperature (RT).

5. Set the wavelength of spectrophotometer at OD 562 nm. Calibrate the instrument to zero by using water. Subsequently, measure the absorbance of all samples within 10 minutes.
Note: Color development continues even after cooling to RT. However, the subsequent development at RT is too weak to produce significant error if all absorbance measurements are made within 10 minute.

6. Subtract OD₅₆₂ of Blank from all readings.

7. Plot the BSA standard curve: OD562 (on Y axis) vs BSA Standard concentration (on X axis). Use the standard curve to determine the protein concentration of each unknown sample.

• Microplate Procedure

1. Add 150 μl of each standard and protein samples into separate microplate wells.

2. Add 150 μl of BCA working reagent to each well and mix thoroughly for 30 seconds.

3. Seal plates and incubate at 37°C for 2 hours or at 60°C for 1 hour.

4. Cool plate to room temperature (RT).

5. Measure the absorbance at 562 nm on a plate reader within 10 minutes.

6. Subtract OD₅₆₂ of Blank from all readings.

7. Plot the BSA standard curve: OD562 (on Y axis) vs BSA Standard concentration (on X axis). Use the standard curve to determine the protein concentration of each unknown sample.