Measuring proteins with the Bradford assay

The first part of this post features the work of José María Espinosa Bernal and Eva Sánchez Escribano. They have developed a protein assay method and collected calibration data using the Open Colorimeter and a 595nm LED. José María and Eva are scientists and educators at I.E.S. Juan Carlos I secondary school in Murcia, Spain. You can follow more of their work where they share great biotechnology educational resources through the website Biotecnología Fácil and on social media @BiotecnologiaF. In the second part of this post we have used their data as an example of how to import calibration data into the Open Colorimeter for regular sample measurements - in this case measuring protein in samples.

Part 1: Bradfords Protein Assay

Authors: José María Espinosa Bernal, Eva María Sánchez Escribano

Principle

The Bradford protein assay is based on the observation that the absorbance maximum for an acidic solution of Coomassie Brilliant Blue G-250 shifts from 465 nm to 595 nm when binding to protein occurs. 

Absorbance spectrum

  • Reported values in literature around 595 nm
  • Measured absorbance peak at 595 nm
  • Open Colorimeter measurement LED:  595 nm
barrido bradford unicam visible uv-uv2doble haz
Hoja 1 Wavelength (nm),A-30 microg protein,A-15 microg protein 480,0,319,0,160 490,0,325,0,163 500,0,341,0,171 510,0,372,0,186 520,0,439,0,220 530,0,553,0,277 540,0,729,0,365 550,0,965,0,483 560,1,204,0,602 570,1,386,0,693 580,1,536,0,768 590,1,587,0,794 600,1,596,0,798 610,1,573,0,787 620,1,470…

Materials

Prepare Bradford calibration solutions

  • Prepare a stock solution of 1 mg/ml BSA  in a 100 mL volumetric flask. Fill the line with distilled water
  • Make a 10-fold dilution of the 1 mg/ml BSA stock for a working 100 µg/ml BSA standard
  • Using the 1.5mL microtubes, prepare the calibration solutions as outlined in the table below

BSA Conc. (µg/ml)

100 µg/ml BSA solution (µL)

Distilled water (µL)

0

0

100

10

10

90

20

20

80

40

40

40

60

60

70

80

80

20

100

100

0

Method

  • To each 100 µl sample, add 1 ml of Bradford reagent
  • Wait at least 5 mins for the color to develop
  • Make sure the Open Colorimeter has the 595 nm LED installed
  • Turn on the Open Colorimeter and select absorbance
  • Blank the colorimeter with distilled water
  • Measure absorbance of all of the solutions
Image of protein calibration solutions
Calibration data

Part 2: Importing protein calibration data to Open Colorimeter

Author: Jo Long

In this section we will import the Bradford protein assay calibration data into the Open Colorimeter so it can be easily accessed to measure protein concentrations in samples. To do this we will use the oc-calibration-app described in this guide:

Creating custom calibrations using oc-calibration-app
In this set of notes we describe how to generate custom calibrations for the Open Colorimeter using a simple web app called the oc-calibration-app. This app makes fitting your calibration data and generating the required .json file for your colorimeter quick and easy. The app is developed using the…

The first step is to save the calibration data in a CSV file. We subtracted the absorbance from the zero concentration sample value from the other samples values in order to produce a plot that passes through (0,0). A file with the data is shown below.

The next step is to fill out the the oc-calibration app fields and upload the calibration .csv file:

Image of oc-calibration-app

The app generates a plot to visually check the calibration data and .json code for the Open Colorimeter firmware.

Plot generated by the oc-calibration-app
{
  "Protein Assay": {
    "units": "ug/mL",
    "led": 595,
    "fit_type": "linear",
    "fit_coef": [
      112.15564695244164,
      0
    ],
    "range": {
      "min": 0,
      "max": 0.7
    }
  }
}

json code generated by the oc-calibration-app

Copy/paste the .json code into the calibrations.json file on the Open Colorimeter and upload to the Open Colorimeter as described in this tutorial:

Installing/upgrading the firmware
In this set of notes we describe how to upgrade or reinstall the firmware on your Open Colorimeter. These instructions can be used whever there is a new firmware release that has new features that you would like to use. There are three steps: * Step One: Download and unzip the

When using this calibration the colorimeter should be blanked with a zero concentration sample containing the Bradford reagent.

The main menu of your Open Colorimeter should now include the Protein Assay as shown in the images below. You can select this method from the menu along with the 595nm LED to measure protein concentrations in your sample using José and Eva's method.

Open Colorimeter Main Menu
Open Colorimeter with the Protein Assay selected

We are grateful to José María Espinosa Bernal and Eva Sánchez Escribano for sharing their work with this community. Please take a moment to check out Biotecnología Fácil to learn more. We have added links to this protein assay and the recent soil active carbon assay in the Open Colorimeter library. We hope to continue building out the library with tests developed and shared by the open hardware community! To share your test, please fill out this form below or email jo@iorodeo.com.
Open Colorimeter Tests
Colorimeters are extremely flexible and easy to use lab instruments. Below are just some examples of how colorimeters are used: * Water quality testing: there are a wealth of colorimetric assays for measuring water parameters such as water turbidity, chlorine content, pH, water hardness, phosphate…