Bradford Protein Assay: Principle, Protocol & Calculations

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  • 0:03 Bradford Protein Assay…
  • 1:09 Standard and Steps
  • 2:12 Sample and Steps
  • 3:14 Example Calculation: Standard
  • 4:01 Example Calculation: Sample
  • 4:54 Lesson Summary
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Lesson Transcript
Instructor
Laura Foist

Laura has a Masters of Science in Food Science and Human Nutrition and has taught college Science.

Expert Contributor
Amanda Robb

Amanda holds a Masters in Science from Tufts Medical School in Cellular and Molecular Physiology. She has taught high school Biology and Physics for 8 years.

The Bradford Protein Assay is one of the methods used to measure protein concentration in a sample. In this lesson we will learn how it works and the steps in this method.

Bradford Protein Assay Principle

How much protein was in the egg you just ate? Or in the Snickers bar you had for a snack? There are several different methods used to measure the protein in food. One of these methods is the Bradford Protein Assay.

The Bradford Protein Assay measures the concentration protein by adding Coomassie dye to the sample under acidic conditions. When proteins bind with the Coomassie dye, the sample changes color from brown to blue. The level of blue can then be measured using a spectrophotometer to determine the concentration of protein in the sample.

Recall that there are 20 amino acids, protein building blocks, in the body. Technically the Bradford Protein Assay is only measuring the basic amino acids, arginine, lysine, and histidine. However, most proteins have a fairly balanced level of these amino acids with all other amino acids, so we can still extrapolate the level of total protein in the sample. But, if a sample is particularly high or low in any of these amino acids, then this is not the right test to use.

Standards and Steps

There are two parts in the Bradford Protein Assay, first the standard and then the sample:

Standard

For the standard, you can use any complete protein, but typically bovine serum albumin (BSA) is used as the standard, since it is cheap and easy to come by.

Step 1: Prepare several dilutions of the BSA standard, at least 5. For example, the dilutions may be 5, 10, 25, 50, 75, and 100 micrograms of BSA per milliliter.

Step 2: Add reagent (which contains an acid and the Coomassie dye) to the BSA dilutions

Step 3: Incubate for 5 min to 1 hour

Step 4: Measure absorbance, with spectrophotometer set at 595 nm

The data obtained here can be used to create a graph, with the absorbance on the y-axis and the known protein concentration on the x-axis. A fairly linear line should be able to be drawn between each of the points, and the equation of the line determined.

Sample

The sample process is very similar to the standard process. The only difference is that we don't know the concentration of the sample. If you have a general idea of about how much protein is in the sample, then dilute it so that it is within the concentration of the standard curve obtained with the BSA analysis. If the sample concentration is completely unknown, then use trial and error. If the absorbance does not fall between 0.2-1 abs, then change the dilution of the sample until the absorbance is within the proper range.

Step 1: Dilute sample so that it falls within the BSA standard curve

Step 2: Add Bradford reagent

Step 3: Incubate for 5 min to 1 hour (as close as possible to how long the BSA was incubated)

Step 4: Measure absorbance, with spectrophotometer set at 595 nm

Plug the absorbance into the equation determined for the standard. The absorbance is plugged in as y, and the protein concentration is determined by calculation x.

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Additional Activities

Bradford Protein Assay Lesson

The best way to learn is to become the teacher yourself. In this lesson, students will pretend to be the teacher and create a short lesson to present to an adult or fellow students. For example, students could create a short slide presentation about what the Bradford protein assay is and how to do it. Then, students would create a worksheet that allows the new students to graph the data and calculate the slope to find the protein concentration, as was shown in the "Sample" section of the lesson. Students can then review their students' work and go over the answers.

Instructions

One of the best ways to master new material is to actually become the teacher yourself. This is exactly what you'll be doing in this activity. Here, you'll be creating a short slide presentation about the Bradford protein assay to use as an educational tool to teach a friend or family member about the assay. Then, you will create a worksheet that asks that person to create a graph from given data, then find the protein concentration in the sample. Then, you'll go over the answers with your "student" and address any misconceptions. To make sure all of the parts of your lesson are complete, check out the criteria for success below.

Criteria for Success

Presentation

  • Presentation explains the principle behind the Bradford protein assay.
  • Presentation covers how to do the assay and the calculations to find the protein concentration.
  • Presentation is colorful and professionally designed.
  • Presenter is knowledgeable, professional and does not read from the slides during the lesson.

Worksheet

  • Worksheet includes at least one sample problem for students to work through that gives students data and asks them to graph and find the protein concentration.
  • Worksheet is professionally designed in a word processing software.

Review

  • Student reviews the correct answers with their audience.
  • Student is patient and knowledgeable and answers any questions from the audience.

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