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 R²=0.987 0.6- ཚཱ ཎྜ རྩ ལྔ 0 0 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 Absorbance A 0.202 B 0.681 C 0.947 D 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 R²=0.987 0.6- ཚཱ ཎྜ རྩ ལྔ 0 0 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 Absorbance A 0.202 B 0.681 C 0.947 D 0.241

Principles of Instrumental Analysis

7th Edition

ISBN:9781305577213

Author:Douglas A. Skoog, F. James Holler, Stanley R. Crouch

Publisher:Douglas A. Skoog, F. James Holler, Stanley R. Crouch

Chapter20: Molecular Mass Spectrometry

Section: Chapter Questions

Problem 20.9QAP

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Instructions You are required to produce a short lab report using the information given in the accompanying document, ‘Lab report assessment_student data’. This document introduces an experiment, and also provides a set of experimental data for this experiment. You are required to use this data to perform steps 8 and 9 of the procedure. This includes calculations of a particular value, and then comparison to the true value. Consider any experimental errors when comparing calculated values and true values. Your report should be between 300 and 500 words (including all table contents and titles of tables and graphs). Your lab report should consist of three sections only: • Experimental • Results and Discussion • Conclusion You do NOT need to include an introduction section. Your experimental section should be written in appropriate lab report format, (passive voice, 3 rd person, in paragraphs). Your results and discussion section should contain the data presented in table format, and graph format, produced using suitable computer software. Your conclusion should state any conclusions which can be drawn based on the experimental data, as detailed in the accompanying document. Submission Guidelines You must submit an electronic copy of your lab report at the submission point on Myplace, by the date and time specified. Late submission will be penalised by an incremental decrease of your grade. Please name your document in the following format and include your registration number on the first page of your report. Please do NOT put your name anywhere on the document.

Summary

Chemistry Lab Report: Protein Concentration Determination

Bradford assay: Determine protein concentration in unknown samples using a standard curve.

Data analysis: Calculate protein concentration in unknown samples using a calibration graph.

Experimental errors: Analyze potential sources of error and their impact on results.

Results and discussion: Present data in tables and graphs, analyze results, and discuss findings.

Conclusion: Draw conclusions about the effectiveness of different protein extraction methods.

Software: Use Excel or similar software for data analysis and graph generation.

Report format: Follow standard lab report format (passive voice, 3rd person).

Word count: Report should be between 300 and 500 words.

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
R²=0.987
0.6-
ཚཱ ཎྜ རྩ ལྔ
0
0
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 Absorbance
A
0.202
B
0.681
C
0.947
D
0.241

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