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Measuring proteins with the Bradford assay

Methods for measuring protein concentration using the Open Colorimeter and 595nm LED.

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

Materials

Open Colorimeter with 595 nm LED board
Bradford reagent and BSA standard
Distilled water
Cuvettes
1.5 mL microtubes
100 mL volumetric flask
100 µL adjustable micropipette

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

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:

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.

Protein Calibration Data - Bradfords Assay

CSV file for generating a .json file for the Open Colorimeter

bradford_calibration_data_blanked.csv

60 Bytes

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

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:

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 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.