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BCA Protein Assay
Product key features
- Rapid results: Completes assay in 30-60 minutes at room temperature.
- Accurate quantitation: Uses proven copper-chelation chemistry.
- Direct equivalent: Replacement of Pierce™ BCA Protein Assay Kit from Thermo Fisher.
- Versatile applications: Suitable for protein quantitation in cell lysates, serum, and other biological fluids.
Product description
Amplite® Colorimetric BCA Protein Quantitation Assay Kit is a colorimetric assay for quantifying total protein concentration in biological samples. It is based on the reduction of Cu²⁺ to Cu⁺ by peptide bonds under alkaline conditions, followed by the chelation of Cu⁺ with bicinchoninic acid (BCA) to form a stable purple complex. The intensity of the color, measured at 562 nm, is directly proportional to protein concentration.
This two-step reaction provides a robust and detergent-compatible assay format. The BCA assay offers a broader detection range and greater stability than traditional Bradford assays, with less variability due to differences in amino acid composition. It is ideal for quantifying proteins in lysates, extracts, or purified preparations, and can be read using a standard microplate reader or spectrophotometer.
Example protocol
AT A GLANCE
Protocol summary
- Prepare BCA working solution (50 µL)
- Add BSA standards or test samples (50 µL)
- Incubate at room temperature for 20 - 60 minutes
- Read absorbance at 562 nm (in the range of 540-590 nm)
Important
Thaw all the kit components at room temperature before use.PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner:
https://www.aatbio.com/tools/serial-dilution/11115
https://www.aatbio.com/tools/serial-dilution/11115
BSA Standard
Add 80 µL of 2 mg/mL BSA Standard (Component C) to 320 µL of PBS (not provided) to generate 400 µg/mL BSA standard solution (BS1). Then use 1:2 serial dilutions in PBS to get serially diluted BSA standards (BS2 - BS7). Note: It is necessary to create a standard curve during each assay.PREPARATION OF WORKING SOLUTION
BCA working solution
- Prepare the amount of BCA working solution needed by mixing 50 parts of BCA Solution A (Component A) with 1 part of BCA Solution B (Component B) (50:1, v/v ratio of Solution A: B).
- Mix well until the BCA working solution is a uniform, light green color.
Note 1 mL BCA working solution is enough for 20 tests.
SAMPLE EXPERIMENTAL PROTOCOL
Table 1.Layout of BSA standards and test samples in a clear bottom 96-well microplate. BS= BSA Standards (BS1 - BS7, 400 to 6.25 µg/mL); BL=Blank Control; TS=Test Samples
Table 2.Reagent composition for each well.
| BS1 | BS1 | TS | TS |
| BS2 | BS2 | ... | ... |
| BS3 | BS3 | ||
| BS4 | BS4 | ||
| BS5 | BS5 | ||
| BS6 | BS6 | ||
| BS7 | BS7 | ||
| BL | BL |
| Well | Volume | Reagent |
| BS1-BS7 | 50 µL | Serial dilutions (400-6.25 µg/mL) |
| BL | 50 µL | PBS |
| TS | 50 µL | Test samples |
- Prepare BSA standards (BS), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2.
- Add 50 µL of BCA working solution to each well of BSA standard, blank control, and test samples to make the total assay volume of 100 µL/well.
- Incubate the reaction at room temperature for 20 to 60 minutes.
- Monitor the absorbance with an absorbance microplate reader at OD 562 nm (in the range of 540 to 590 nm).
Alternative formats
References
View all 15 references: Citation Explorer
Specialized Cell-Free DNA Blood Collection Tubes Can Be Repurposed for Extracellular Vesicle Isolation: A Pilot Study.
Authors: Heatlie, Jessica and Chang, Vanessa and Fitzgerald, Sandra and Nursalim, Yohanes and Parker, Kate and Lawrence, Ben and Print, Cristin G and Blenkiron, Cherie
Journal: Biopreservation and biobanking (2020)
Authors: Heatlie, Jessica and Chang, Vanessa and Fitzgerald, Sandra and Nursalim, Yohanes and Parker, Kate and Lawrence, Ben and Print, Cristin G and Blenkiron, Cherie
Journal: Biopreservation and biobanking (2020)
Protein Quantitation and Analysis of Purity.
Authors: Campion, Eva M and Loughran, Sinéad T and Walls, Dermot
Journal: Methods in molecular biology (Clifton, N.J.) (2017): 225-255
Authors: Campion, Eva M and Loughran, Sinéad T and Walls, Dermot
Journal: Methods in molecular biology (Clifton, N.J.) (2017): 225-255
Proteomic Approach for Extracting Cytoplasmic Proteins from Streptococcus sanguinis using Mass Spectrometry.
Authors: El-Rami, Fadi and Nelson, Kristina and Xu, Ping
Journal: Journal of molecular biology research (2017): 50-57
Authors: El-Rami, Fadi and Nelson, Kristina and Xu, Ping
Journal: Journal of molecular biology research (2017): 50-57
[iTRAQ technology combined with 2D-LC-MS/MS to analyze effect of Coptidis Rhizoma on cytochrome P450 isoenzyme expression].
Authors: Yang, Xin and Wang, Qiu-Hong and Wang, Meng and Wang, Yue and Yang, Bing-You and Kuang, Hai-Xue
Journal: Zhongguo Zhong yao za zhi = Zhongguo zhongyao zazhi = China journal of Chinese materia medica (2016): 731-736
Authors: Yang, Xin and Wang, Qiu-Hong and Wang, Meng and Wang, Yue and Yang, Bing-You and Kuang, Hai-Xue
Journal: Zhongguo Zhong yao za zhi = Zhongguo zhongyao zazhi = China journal of Chinese materia medica (2016): 731-736
Total protein quantitation using the bicinchoninic acid assay and gradient elution moving boundary electrophoresis.
Authors: Kralj, Jason G and Munson, Matthew S and Ross, David
Journal: Electrophoresis (2014): 1887-92
Authors: Kralj, Jason G and Munson, Matthew S and Ross, David
Journal: Electrophoresis (2014): 1887-92
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