Communication pubs.acs.org/jchemeduc
Quantitative Application for SDS−PAGE in a Biochemistry Lab John Carter, Brandon P. Petersen, Sarah A. Printz, Timothy L. Sorey, and Todd T. Kroll* Department of Chemistry, Central Washington University, Ellensburg, Washington 98926-7539, United States
J. Chem. Educ. 2013.90:1255-1256. Downloaded from pubs.acs.org by PURDUE UNIV on 10/28/18. For personal use only.
S Supporting Information *
ABSTRACT: A variety of laboratory experiments have been published in this Journal describing the use of polyacrylamide gel electrophoresis to investigate different properties of proteins, including determination of the subunit composition of individual proteins and molar mass. The experiment described extends the capabilities of this experimental technique and provides a mechanism to determine the concentration of a single protein from a heterogeneous mixture.
KEYWORDS: Upper-Division Undergraduate, Biochemistry, Laboratory Instruction, Hands-On Learning/Manipulatives, Electrophoresis, Quantitative Analysis
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with a common desktop scanner, and the resulting image is imported into the freeware program ImageJ (NIH) for analytical analysis (Figure 1).
olyacrylamide gel electrophoresis (PAGE) is commonly conducted in the presence of sodium dodecyl sulfate (SDS−PAGE) to resolve polypeptides for two types of analysis: (i) to determine the molecular mass of proteins and (ii) to analyze the subunit composition of a given protein.1,2 An often overlooked capability of SDS−PAGE is that it can be used to quantitate the concentration of a single protein from a heterogeneous mixture of proteins, which is commonly accomplished by ELISA (enzyme-linked immunosorbant assay) or Western blotting. The experiment described, which is amenable to modification for a wide range of applications, combines the generation of a standard curve of bovine serum albumin (BSA), as described by Barufaldi et al.,3 with SDS− PAGE to determine the concentration of a single protein, serum albumin, from a sample of whole human serum. This experiment provides an economical method for determining the concentration of albumin in a sample of commercially available human serum by utilizing a common desktop scanner and free software (ImageJ) from the National Institutes of Health,4 and is readily adapted to determine the concentration of any protein of interest from a heterogeneous mixture.5
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DISCUSSION This laboratory experiment involved students running multiple BSA samples, prepared by serial dilution, to quantify the concentration of a particular protein from a mixture without
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Figure 1. Scanned image of an SDS−polyacrylamide gel, dried between sheets of cellophane, produced by a group of students. Lanes 1, 2, and 4 contain 0.125, 0.25, and 0.5 μL of human serum. Lane 3 is empty. Lane 5 contains prestained molecular mass markers (Precision Plus Dual Color Protein Standards, Bio-Rad). Lanes 6−10 contain 1, 2, 4, 8, and 16 μg of BSA protein. The densitometric scan below the gel represents the color density within the bracketed region (the scan of the marker lane has been deleted for clarity); the numbers below the scans represent the integrated color densities for the peaks.
EXPERIMENT Students generate a standard curve of BSA, prepared by serial dilution, ranging from 0.1 to 1.6 μg/μL, from a stock solution of 10 mg/mL BSA. Students are also provided with diluted human serum (Sigma Aldrich) of unknown concentration, molecular mass markers, a polyacrylamide gel, and electrophoresis equipment and buffers. Following sample preparation and separation by electrophoresis, the proteins are stained with Coomassie blue. The gels are destained to eliminate background, dried between two sheets of cellophane, and scanned © 2013 American Chemical Society and Division of Chemical Education, Inc.
Published: July 24, 2013 1255
dx.doi.org/10.1021/ed300390j | J. Chem. Educ. 2013, 90, 1255−1256
Journal of Chemical Education
Communication
(4) ImageJ, 1.46; National Institutes of Health: Bethesda, MD, 2012. http://rsbweb.nih.gov/ij/download.html (accessed Jun 2013) (5) Kubo, Y.; Takenaka, H.; Nagai, H.; Toyokuni, S. J. Clin. Biochem. Nutr. 2012, 51, 221−226. (6) Pratt, D. S. Liver chemistry and function tests. In Sleisenger and Fordtran′s Gastrointestinal and Liver Disease; Feldman, M., Friedman, L. S., Brandt, L. J., Eds.; Elsevier: Philadelphia, 2010: Chapter 73. (7) Crispell, K. R.; Porter, B.; Nieset, R. T. J. Clin. Invest. 1950, 29, 513−516.
using expensive antibodies, equipment, or software. This exercise required approximately three hours in the laboratory and an additional one to two hours for computer analysis and reinforced the concept of standard curves and acted as an introduction to protein analysis. All groups of third- and fourthyear undergraduate students (trials included over 100 students) who performed the experiment without major error obtained R2 values of >0.95. Application of the standard curve in Figure 2
Figure 2. Standard curve for the BSA. Data were obtained from student analysis of the gel shown in Figure 1.
provided final concentrations of human serum albumin of 4.7, 5.4, and 4.8 g/dL, when 0.125, 0.250, and 0.50 μL of human serum were analyzed, respectively. This was reliable; when the line equation was applied to samples of human serum albumin that fell within the standard curve, most students acquired values within the accepted range of 3.4−5.4 g/dL.6 Using this value, a student can calculate the total mass of albumin in a typical human when given the average human serum volume of 2.7−3.0 L and the molecular mass of the human albumin.7 The calculations performed in this exercise could easily be incorporated into laboratories qualitatively analyzing subunit composition of proteins to include the quantitative determination of stoichiometric composition.2 Laboratories equipped with a gel-doc station may use a photograph of the wet gel for analysis, although this provided significantly reduced R2 values in our side-by-side comparisons.
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ASSOCIATED CONTENT
S Supporting Information *
Instructions for students and instructors, experimental procedures, hazards information, CAS numbers, recipes, and a grading rubric. This material is available via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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REFERENCES
(1) Nash, B. T. J. Chem. Educ. 2007, 84, 1508−1510. (2) Powers, J. L.; Andrews, C. S.; St. Antoine, C. C.; Jain, S. S.; Bevilacqua, V. L. J. Chem. Educ. 2005, 82, 93−95. (3) Barufaldi, M.; Pappano, N. B.; Debattista, N. B. J. Chem. Educ. 1999, 76, 965−966. 1256
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