Feb 01, 2025

Public workspacePierce BCA Protein Assay Protocol

 Forked from Pierce BCA Protein Assay Protocol
DOI
dx.doi.org/10.17504/protocols.io.x54v9rod4v3e/v1
  • 1Ph.D.;
  • 2Emory University
Alexandria White
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DOI: dx.doi.org/10.17504/protocols.io.x54v9rod4v3e/v1
External link: https://doi.org/10.1371/journal.pone.0190546
Protocol Citation: James P. O’Callaghan, Alexandria White 2025. Pierce BCA Protein Assay Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9rod4v3e/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: January 31, 2025
Last Modified: February 01, 2025
Protocol Integer ID: 119370
Funders Acknowledgements:
Aligning Science Against Parkinson's (ASAP)
Grant ID: Grant ID: ASAP-020527
Abstract
This is the protocol to be used in the determination of total protein in tissue by the Pierce BCA method. BSA (Bovine Serum Albumin) is used as the standard and samples are prepared by sonification in 1% homogenization buffer, the recipe for which is provided in the protocol below.
Guidelines
INTRODUCTION
The Thermo Scientific™ Pierce™ BCA Protein Assay is a detergent-compatible formulation based on bicinchoninic acid (BCA) for the colorimetric detection and quantitation of total protein. This method combines the well-known reduction of Cu+2 to Cu+1 by protein in an alkaline medium (the biuret reaction) with the highly sensitive and selective colorimetric detection of the cuprous cation (Cu+1) 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 562nm that is nearly linear with increasing protein concentrations over a broad working range (20-2000μg/mL). The BCA method is not a true end-point method; that is, the final color continues to develop. However, following incubation, the rate of continued color development is sufficiently slow to allow large numbers of samples to be assayed together.
The macromolecular structure of protein, the number of peptide bonds and the presence of four particular amino acids (cysteine, cystine, tryptophan and tyrosine) are reported to be responsible for color formation with BCA. Studies with di-, tri- and tetrapeptides suggest that the extent of color formation caused by more than the mere sum of individual color-producing functional groups. Accordingly, 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 unknown(s) before the concentration of each unknown is determined based on the standard curve. If precise quantitation of an unknown protein is required, it is advisable to select a protein
standard that is similar in quality to the unknown; for example, a bovine gamma globulin (BGG) standard may be used when assaying immunoglobulin samples.
TRAINING REQUIREMENTS
Able to use a plate reader and to accurately dilute tissue samples.
REFERENCES
BCA Protein Assay Reagent (instruction manual), Pierce. Rockford, IL.
Softmax User’s Manual, Molecular Devices Corporation. Sunnyvale, CA.
Materials
MATERIALS
ReagentPlate Reader
ReagentPipettes
ReagentIncubator
ReagentVortex Mixer
ReagentMicrotubes 1.2 ml
Reagent96 well microtiter plates
ReagentPipette tips
ReagentPierce BCA Protein Assay Kit Thermo Fisher ScientificCatalog #23225
ReagentPierce BCA Protein Assay Reagent A Thermo Fisher ScientificCatalog #23221
ReagentPierce BCA Protein Assay Reagent B Thermo Fisher ScientificCatalog #23224
ReagentBovine Serum Albumin (BSA) Sigma AldrichCatalog #A7906
ReagentSodium Dodecyl Sulfate Bio-rad LaboratoriesCatalog #1610416
Safety warnings
CAUTIONARY NOTES/SPECIAL CONSIDERATIONS
BCA reagent is stable for up to one week; standard practice dictates the use of 50ml quantities, enough to do two full microtiter plates.
SDS serves as a wetting agent and pipetting SDS-containing samples can lead to pipetting errors. Therefore, use only a single pipette tip per sample and withdraw the sample only a single time per tip. This procedure holds for SDS and all SDS-containing samples.
REAGENT PREPARATION
REAGENT PREPARATION
1.   1% Homogenization Buffer (HB)
Prepare a solution of 1.0 M Tris, 0.5 M MgCl2, 0.1 M EDTA, deionized water, and 1% Triton-X 100 with protease and phosphatase inhibitors (Pierce).
2.   BCA Reagent
This reagent is made according to the directions provided by Pierce, 50ml of solution A and 1ml of solution B. Thoroughly mix while adding solution B.
STANDARD PREPARATION
STANDARD PREPARATION
Protein Standards: Bovine Serum Albumin (BSA), modified for use in strip tubes.
1.  For this step, commercial albumin standard is used (Thermo, Ref 23209) to create the BSA standard curve.
2.   Aliquot as needed and store at -80C for long-term storage or 4C if to be used within 2 weeks.
3. Create standards according to the following table:
ABCD
TubeHB (uL)BSA (uL)Concentration (ug/mL)
A0100 stock2000
B42125 stock1500
C110110 stock1000
D6060 of B750
E110110 of C500
F110110 of E250
G110110 of F125
H13535 of G25
I13500

4. Vortex each tube of the diluted BSA standard & remove 25µl of the diluted standard and place it in the designated well on a 96-well plate in duplicates.
SAMPLE PREPARATION
SAMPLE PREPARATION
The unknown experimental samples are 10 fold.
1.   Remove samples from the freezer and allow to thaw at room temperature.
2.   Vortex and then remove a 10µl aliquot from the sample and place it into a dilution tube. Add 90µl of 1% HB to each sample tube.

3.   Vortex the dilution tubes and remove a 10µl aliquot and place it in the appropriate well on the microtiter plate, in duplicate.
4.   Repeat steps 1 through 3 for each experimental sample.
PROTEIN ASSAY
PROTEIN ASSAY
1.   Add 200µl of pre-mixed working reagent to each of the wells containing either the standard curve or sample. Mix plate gently on a shaker.

2.   Incubate the plate at 37°C for 30 minutes in the dark.
3.   At the completion of the incubation period, the plate is checked for bubbles and then read in the plate reader at a wavelength of 562nm. “Pop” any bubbles with a needle or a pipette tip prior to reading the plate.
CALCULATIONS
CALCULATIONS
1.   Software programs coupled to specific plate readers are used to calculate protein values based on comparing OD readings of the unknowns with those obtained from the standard curve. OD values and standard protein values are plotted in a linear fashion on both axes. The following is a typical standard curve for the BCA Protein Assay when bovine serum albumin is used for the standard. The table gives the actual values for each point on the standard curve and their corresponding OD readings.
  BCA Total Protein Standard Curve    

Plate Blank Well OD Mean Std Dev CV
BL A1 0.083 0.082 0.001 1.725
  A2 0.081      
STANDARD Value Well  OD Mean Std Dev  CV
STD01 1.000   µg A3 0.06 0.058 0.002 3.626
    A4 0.057      
             
STD02 2.500   µg A5 0.131 0.131 7.07E-04 0.542
    A6 0.13      
             
STD03 5.000   µg A7 0.22 0.218 0.003 1.297
    A8 0.216      
             
STD04 7.500   µg A9 0.311 0.321 0.014 4.406
    A10 0.331      
             
STD05 10.00   µg A11 0.419 0.418 0.001 0.338
    A12 0.417      
2.   The blank can be automatically subtracted from all other OD readings or it must be done manually if this feature is not available.
3.   The dilution factors can be entered into the programs, otherwise they must be considered in the final calculations.
4.   The final printout will include a plot of the standard curve and the protein concentration of each unknown expressed as mg/ml or µg/µl. The table below gives the calculated protein values for unknown hippocampus samples in mg/ml or µg/µl. These values include the subtraction of the blank value and a dilution factor of 2.
UNKNOWN Mean Std Dev  CV Well  OD Value Dil. Factor
UNK01 9.325 0.215 2.306 B1 0.211 9.477 2
        B2 0.205 9.173  
               
UNK02 10.44 ***** ***** B3 0.23 10.44 2
        B4 0.23 10.44  
               
UNK03 9.883 0.072 0.725 B5 0.218 9.832 2
        B6 0.22 9.934  
               
UNK04 11.18 1.254 11.23 B7 0.227 10.29 2
        B8 0.262 12.06  
               
UNK05 11.45 0.717 6.258 B9 0.24 10.95 2
        B10 0.26 11.96  
5.   Example of calculations, if samples are prepared as listed above:
                  a.   Hippo#3 is prepared as a 1:20 dilution (10µl of standard or unkown sample are placed in a dilution tube and brought up to a final volume of 200µl)
                  b.   OD reading for this sample is 0.219
                  c.   OD of .219 corresponds to » 4.942µg/10µl or 0.4942µg/µl total protein        
                  d.   This sample has been diluted 1:20 so the value is actually 0.4942µg/µl x 20= 9.883µg/µl (since the protein sample was loaded in 10 µl volumes the value can be determined by using a dilution
                        factor of 2, That is, 4.942µg/10µl x2=9.883µg/µl).