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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.

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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