Apr 22, 2026
Pierce™ BCA Protein Assay (ThermoFisher Scientific)
- Rita Chen1,
- Raven Coffer1
- 1DAMP Lab
- DAMP Lab
Protocol Citation: Rita Chen, Raven Coffer 2026. Pierce™ BCA Protein Assay (ThermoFisher Scientific). protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2zp5jl1y/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 19, 2026
Last Modified: April 22, 2026
Protocol Integer ID: 243691
Keywords: thermofisher scientific pierce bca protein assay kit, bca protein assay, common protein such as bovine serum albumin, bovine serum albumin, protein quantification, molecules of bca, protein in an alkaline medium, bicinchoninic acid, containing bicinchoninic acid, selective colorimetric detection of the cuprous cation, reaction product of this assay, protein concentration, protein, assay, common protein, increasing protein concentration, reduction of cu2, selective colorimetric detection, colored reaction product, bca, cu2, chelation, alkaline medium, strong absorbance, cu1, biuret reaction
Abstract
The protocol uses the ThermoFisher Scientific Pierce BCA Protein Assay kit (CAT #23225) to perform protein quantification. This method combines the well-known reduction of Cu2+ to Cu1+ by protein in an alkaline medium (the biuret reaction) with the highly sensitive and selective colorimetric detection of the cuprous cation (Cu1+) using a unique reagent containing bicinchoninic acid. The purple-colored reaction product of this assay is formed by the chelation of two molecules of BCA with one cuprous ion. This water-soluble complex exhibits a strong absorbance at 562 nm that is nearly linear with increasing protein concentrations over a broad working range (20–2000 µg/mL). Protein concentrations generally are determined and reported with reference to standards of a common protein such as bovine serum albumin (BSA). A series of dilutions of known concentration are prepared from the protein and assayed alongside the unknowns before the concentration of each unknown is determined based on the standard curve.
Image Attribution
^ From ThermoFisher
Materials
Consumables
- 96-well Clear Flat-bottomed plate
- semi-permeable 96-well plate seal
Kit-Specific
- Pierce BCA Protein Assay Reagent A
- Pierce BCA Protein Assay Reagent B
- Pierce BSA (BSA) Protein Standards (tubestrip)
Equipment
- Synergy H1 Microplate Reader
Overview
This is a slightly modified version of the Pierce™ BCA Protein Assay (ThermoFisher Scientific).
Figure 1: Graphical depiction of procedure summary, from ThermoScientific
Storage
| Component | Storage Temperature | |
| BCA Assay Reagent A | 15 - 30 C | |
| BCA Assay Reagent B | 15 - 30 C | |
| BSA Protein Standards | 15 - 30 C (4C if previously opened) |
Table 1: Required reagents and respective storage temperature
Reagent Preparation
13m
Preparation of Standards
In 2 mL tubes, make nine standards in a working range of 25-2,000 µg/mL from the formulas in the following table. These will be used to make a standard curve for linear regression later in the experiment.
| Vial | Volume of Diluent (µL) | Volume and Source of BSA (µL) | Final BSA Conc. (µg/mL) | |
| A | 0 | 300 µL of stock | 2,000 | |
| B | 125 | 375 µL of stock | 1,500 | |
| C | 325 | 325 µL of stock | 1,000 | |
| D | 175 | 175 µL of vial B | 750 | |
| E | 325 | 325 µL of vial C | 500 | |
| F | 325 | 325 µL of vial E | 250 | |
| G | 325 | 325 µL of vial F | 125 | |
| H | 400 | 100 µL of Vial G | 25 | |
| I | 400 | 0 µL | (Blank) |
Table 2: Dilution volumes for standard samples
Preparation of Working Reagent (WR)
3m
Download this Excel spreadsheet and enter the number of unknown samples in cell B6 to calculate your total volume of WR. It will also calculate your required volumes of Reagent A and Reagent B to achieve a 20:1 concentration of Reagent A to Reagent B.
BCA Protein Assay Calculation Template.xlsx Note
In this protocol, the assay is performed in triplicates. If more replicates are desired, the modify the spreadsheet so your number of replicates, samples, and unknowns are accurate.
Note
This spreadsheet will be used later in this protocol to calculate sample absorbance and perform a linear regression to calculate the concentrations of your unknown samples.
Protein Assay
50m
Prepare Microplate
To create a 1:8 ratio of sample:WR ...
- Add 200 µL of the prepared WR to each well
- Add 25 µL of each sample in triplicate
- Pipette 10X per well to mix.
Note
Each well has a final volume of 225 µL .
5m
Cover the plate with a semi-permeable seal, and incubate at 37 °C static for 00:30:00 .
30m
With the seal on, cool the plate to Room temperature for 00:10:00 .
10m
Plate Reader Assay
5m
Measure Sample Absorbance
Remove the seal and dispose in biohazard. Measure the absorbance at 562 nm on the BioTek Synergy H1 microplate reader.
Note
Wavelengths from 540–590 nm have been used successfully with this method.
Input the absorbance data into the BCA Protein Assay Calculations Template. Add more unknown rows if needed. The spreadsheet will normalize your data based off the background noise measured in your blanks and perform a linear regression on the absorbance measurements of your unknown samples to find the concentrations.
Note
- If the assay was performed in more replicates, add additional columns and modify the formulas as to include your additional absorbance data. Adding a column between existing replicate columns will update your formulas automatically.
- If the assay was performed with more unknowns, insert additional rows between the preexisting unknown rows to keep your formulas inclusive of all your data.
Cleanup
5m
Empty benchtop sharps container into larger floor sharps container, and store materials at their designated temperature.
Dispose of the 96w assay plate in the biohazard waste bin.
Protocol references
Thermofisher Guide for Pierce‱ BCA Protein Assay Kit with Dilution-Free‱ BSA Protein Standards, Multichannel Pipette Compatible: https://documents.thermofisher.com/TFS-Assets/LSG/manuals/MAN0029423-BCAProteinWithDIlutionFreeBSA-UG.pdf
Thermoscientific Protein Assay Technical Handbook: https://documents.thermofisher.com/TFS-Assets/LSG/brochures/protein-assay-technical-handook.pdf
Acknowledgements
Drafted by: Raven Coffer
Uploaded by: Raven Coffer
Edited by: Raven Coffer, Owen Butler, Lena Landaverde, Kristen Sheldon, Molly Brennan