Determination of protein concentration using the Bradford assay Introduction It is often necessary to determine the concentration of protein in a sample before processing the sample for further analysis. There are several different techniques for measuring protein concentration such as UV absorbance, Biuret methods, colorimetric dye-based methods and fluorescent dye methods. The Bradford assay is a colorimetric protein assay based on the absorbance shift of the dye Coomassie Blue when bound to protein. In the absence of protein, Coomassie is a brown/red colour and when protein is present it turns blue. This increases the absorbance of the sample, which can be measured on a spectrophotometer at the maximum absorbance frequency of the blue dye (595 nm). A higher absorbance means a greater protein concentration. Note, the units of absorbance are known as arbitrary units (AU). To calculate the protein concentration in an unknown sample, a set of standards must be prepared and run using the Bradford assay. Standards are samples with a known amount of protein in them. The absorbance of these standards can be used to produce a calibration graph with protein concentration on the x-axis and absorbance on the y-axis, resulting in a straight line with the equation y = mx + c. An example graph can be seen in figure 1 below. The equation of the straight line from the calibration graph can be used to determine the protein concentration of an unknown sample, based on its measured absorbance. A scientific company has been developing new ways to extract protein. Four samples have been generated for testing, each using a different method of protein extraction (Method A, B, C and D). To determine the most effective method of protein extraction, the protein concentration of each sample must be determined. The most effective extraction method will result in the sample with the highest protein concentration. 1.2 1 y=0.5176x+0.0041 mm 0.8 R0.987 Absorbanc 595 0.4 20.2 0.25 0.5 0.75 1 1.25 1.5 1,75 2 2.25 Figure 1 BSA standard (mg/ml) Example standard curve for Bradford Assay Aim Create a calibration graph (standard curve) using the standard solutions, determine the protein concentration in each of the samples (A, B, C and D) and recommend the best extraction method for the company (A, B, C or D). Experimental 1. Prepare six dilutions of a BSA protein standard with a range of 5-100 µg/ml protein. The highest concentration should be 100 µg/ml and the lowest concentration should be 6.25 µg/ml. 2. Add 30 µl of each standard solution or unknown sample to an appropriately labelled test tube. 3. Add 1 ml of Coomassie Blue to each tube. Mix the sample and Coomassie Blue together using a vortex mixer. 4. Set a timer and incubate the test tubes at room temperature for 5 minutes. 5. Turn on the spectrophotometer and set the absorbance to 595 nm. 6. Place one test tubes in the spectrophotometer sample holder and measure the absorbance. 7. Repeat step 6 until each standard, and each unknown sample have been measured. 8. Using Excel, or similar software, plot the absorbances of the standards and generate a calibration graph, using an xy-scatter graph. To do this the concentrations of the standards should be on the x-axis and the absorbances on the y-axis of the xy-scatter graph. Add a linear trendline and the equation of the line. 9. Using the absorbances and equation of the line, determine the protein concentrations of the unknown samples A to D. Results Standards Concentration Absorbance 100 1.104 75 0.854 50 0.703 25 0.425 12.5 0.241 6.25 0.101 Sample A Absorbance B 0.202 C 0.681 D 0.947 0.241
Determination of protein concentration using the Bradford assay
Introduction
It is often necessary to determine the concentration of protein in a sample before processing the sample for further analysis. There are several different techniques for measuring protein concentration such as UV absorbance, Biuret methods, colorimetric dye-based methods and fluorescent dye methods.
The Bradford assay is a colorimetric protein assay based on the absorbance shift of the dye Coomassie Blue when bound to protein. In the absence of protein, Coomassie is a brown/red colour and when protein is present it turns blue. This increases the absorbance of the sample, which can be measured on a spectrophotometer at the maximum absorbance frequency of the blue dye (595 nm). A higher absorbance means a greater protein concentration. Note, the units of absorbance are known as arbitrary units (AU).
To calculate the protein concentration in an unknown sample, a set of standards must be prepared and run using the Bradford assay. Standards are samples with a known amount of protein in them. The absorbance of these standards can be used to produce a calibration graph with protein concentration on the x-axis and absorbance on the y-axis, resulting in a straight line with the equation y = mx + c. An example graph can be seen in figure 1 below.
The equation of the straight line from the calibration graph can be used to determine the protein concentration of an unknown sample, based on its measured absorbance.
A scientific company has been developing new ways to extract protein. Four samples have been generated for testing, each using a different method of protein extraction (Method A, B, C and D). To determine the most effective method of protein extraction, the protein concentration of each sample must be determined. The most effective extraction method will result in the sample with the highest protein concentration.
1.2
1
y=0.5176x+0.0041
mm
0.8
R0.987
Absorbanc 595
0.4
20.2
0.25
0.5
0.75
1
1.25 1.5
1,75
2
2.25
Figure 1
BSA standard (mg/ml)
Example standard curve for Bradford Assay
Aim
Create a calibration graph (standard curve) using the standard solutions, determine the protein concentration in each of the samples (A, B, C and D) and recommend the best extraction method for the company (A, B, C or D).
Experimental
1. Prepare six dilutions of a BSA protein standard with a range of 5-100 µg/ml protein. The highest concentration should be 100 µg/ml and the lowest concentration should be 6.25 µg/ml.
2. Add 30 µl of each standard solution or unknown sample to an appropriately labelled test tube.
3. Add 1 ml of Coomassie Blue to each tube. Mix the sample and Coomassie Blue together using a vortex mixer.
4. Set a timer and incubate the test tubes at room temperature for 5 minutes.
5. Turn on the spectrophotometer and set the absorbance to 595 nm.
6. Place one test tubes in the spectrophotometer sample holder and measure the absorbance.
7. Repeat step 6 until each standard, and each unknown sample have been measured.
8. Using Excel, or similar software, plot the absorbances of the standards and generate a calibration graph, using an xy-scatter graph. To do this the concentrations of the standards should be on the x-axis and the absorbances on the y-axis of the xy-scatter graph. Add a linear trendline and the equation of the line.
9. Using the absorbances and equation of the line, determine the protein concentrations of the unknown samples A to D.
Results
Standards
Concentration
Absorbance
100
1.104
75
0.854
50
0.703
25
0.425
12.5
0.241
6.25
0.101
Sample
A
Absorbance
B
0.202
C
0.681
D
0.947
0.241
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