Mar 14, 2017
Bradford Protein Concentration determination
- Brian Baltzar
Protocol Citation: Brian Baltzar 2017. Bradford Protein Concentration determination. protocols.io https://dx.doi.org/10.17504/protocols.io.girbud6
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
Created: November 25, 2016
Last Modified: March 19, 2018
Protocol Integer ID: 4401
Keywords: bradford protein concentration determination, bradford protein, protein concentration, assay, concentration
Abstract
Estimate a protein concentration in a solution using the Bradford protein assay
Materials
MATERIALS
Bovine Serum Albumin (IgG-Free, Protease-Free)Jackson ImmunoResearch Laboratories, Inc.Catalog #001-000-161
Protein AssayBio-Rad LaboratoriesCatalog #5000002
Troubleshooting
Make BSA standards
Dilute BSA using the following table.
| Tube | µl BSA (1mg/ml) | µl Buffer | Protein concentration (µg/ml) | |
| 1 | 0 | 30 | 0 | |
| 2 | 0 | 30 | 0 | |
| 3 | 0.5 | 29.5 | 16.67 | |
| 4 | 1 | 29 | 33.33 | |
| 5 | 2 | 28 | 66.67 | |
| 6 | 3.5 | 26.5 | 116.66 | |
| 7 | 5 | 25 | 166.67 | |
| 8 | 10 | 20 | 333.33 | |
| 9 | 15 | 15 | 500 | |
| 10 | 20 | 10 | 666.67 | |
| 11 | 25 | 5 | 833.33 | |
| 12 | 30 | 0 | 1000 |
Dilute Bradford (BioRad) Protein Assay 5X
Add 1.4 mL diluted Bradford (BioRad) Protein assay mix to each of the diluted BSA samples
Add 1.4 mL diluted Bradford (BioRad) Protein assay mix to tubes containing 30µl of the protein of interest.
Vortex for 10 s.
Incubate for 30 min. at room temperature
(Leave no longer than one hour)
Measure the absorbance at 595nm
Plot the OD of the BSA protein standards
(Concentrations at X and OD at Y)
Make a linear regression
Calculate the concentration of the protein of interest.