Amplite® Colorimetric BCA Protein Quantitation Assay Kit

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H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
UNSPSC	12171501

Overview SDS Protocol

The traditional BCA protein assay is widely used for quantifying proteins. However, it is slow and tedious, e.g., you must either heat the BCA reaction at 37 °C for 30 minutes or wait for two hours at room temperature. Amplite®™ Colorimetric BCA Protein Quantitation Assay Kit is a two-component and detergent-compatible assay to determine total protein concentrations. The assay is based on the same copper-chelating reaction and provides comparable accuracy to the traditional BCA protein assay that is either run at high temperature or with longer incubation time. The protein signal is monitored around 560 nm and the assay is completed within 30 minutes. It is convenient, rapid, and robust without high temperature required. Amplite®™ Colorimetric BCA Protein Quantitation Assay Kit can be performed in a convenient 96-well microtiter-plate format and easily adapted to automation with no separation steps required.

Platform

Absorbance microplate reader

Absorbance	562 nm
Recommended plate	Clear bottom

Components

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

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

BS1	BS1	TS	TS
BS2	BS2	...	...
BS3	BS3
BS4	BS4
BS5	BS5
BS6	BS6
BS7	BS7
BL	BL

Table 2.Reagent composition for each well.

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).

Images

Figure 1. . BSA dose responses were measured with Amplite® Colorimetric BCA Protein Quantitation Assay Kit using a clear bottom 96-well plate.

Citations

View all 1 citations: Citation Explorer

Increased alcohol dehydrogenase 1 activity promotes longevity
Authors: Ghaddar, Abbas and Mony, Vinod K and Mishra, Swarup and Berhanu, Samuel and Johnson, James C and Enriquez-Hesles, Elisa and Harrison, Emma and Patel, Aaroh and Horak, Mary Kate and Smith, Jeffrey S and others,
Journal: Current Biology (2023)

References

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Journal: Biopreservation and biobanking (2020)

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

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Journal: Methods in molecular biology (Clifton, N.J.) (2017): 225-255

[iTRAQ technology combined with 2D-LC-MS/MS to analyze effect of Coptidis Rhizoma on cytochrome P450 isoenzyme expression].
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Journal: Zhongguo Zhong yao za zhi = Zhongguo zhongyao zazhi = China journal of Chinese materia medica (2016): 731-736

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Journal: Electrophoresis (2014): 1887-92

Quantitative evaluation of proteins with bicinchoninic acid (BCA): resonance Raman and surface-enhanced resonance Raman scattering-based methods.
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Journal: The Analyst (2012): 5834-8

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Journal: Methods in enzymology (2009): 73-95

Interferences of suspended clay fraction in protein quantitation by several determination methods.
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Journal: Analytical biochemistry (2008): 108-14

A comparison of protein quantitation assays for biopharmaceutical applications.
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