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Section 5 : Protein Studies

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Abstract:

Protein analysis, necessary for an understanding of protein function, often begins with quantitative measurement of total protein concentration in a biological sample. Three different methods are frequently employed in many laboratories. These are the Lowry method, the Bradford method, and the bicinchoninic acid (BCA) method. All three of these methods are sensitive to the amino acid composition of the protein in question and make it difficult to obtain absolute concentration. However, these methods are so widely used and relatively simple to perform that they have become well accepted as means to determine protein concentration, especially in protein mixtures or crude extracts. In each of the aforementioned methods, protein concentration is determined by comparison of unknown samples with a standard curve generated by performing the desired protocol using known concentrations of a protein standard solution. Bovine serum albumin (BSA), fraction V, and bovine gamma globulin (BGG) are the most widely used protein assay standards. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) denatures proteins and separates them based on molecular weight. Coomassie blue staining easily stains all proteins in an acrylamide gel for visualization. SDS-PAGE may be used to analyze cell- and tissue-specific protein expression. For identification of a particular protein resolved by SDS-PAGE, the proteins are transferred out of the gel and immobilized onto a nitrocellulose membrane in a process called Western blotting. Antibodies are used to detect specific proteins in a Western blot. These antibodies are generated by immunizing an animal with the protein of interest.

Citation: Scheppler J, Cassin P, Gambier R. 2000. Protein Studies, p 157-185. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch5

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Figures

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Figure 18.1

The globular structure of native proteins is dissociated by LSB components and heat treatment.

Citation: Scheppler J, Cassin P, Gambier R. 2000. Protein Studies, p 157-185. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch5
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Figure 18.2

Assembly of gel apparatus. (Reprinted from Bio-Rad Laboratories, Hercules, Calif., with permission.)

Citation: Scheppler J, Cassin P, Gambier R. 2000. Protein Studies, p 157-185. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch5
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Citation: Scheppler J, Cassin P, Gambier R. 2000. Protein Studies, p 157-185. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch5
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Figure 19.1

Overview of Western blotting.

Citation: Scheppler J, Cassin P, Gambier R. 2000. Protein Studies, p 157-185. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch5
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Figure 19.2

A direct immunoassay utilizes a tag on the primary antibody, the antibody that binds to the antigen. An indirect immunoassay utilizes a tag on the secondary antibody, which binds to the primary antibody.

Citation: Scheppler J, Cassin P, Gambier R. 2000. Protein Studies, p 157-185. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch5
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Figure 19.3

Assembly of gel apparatus. (Reprinted from Bio-Rad Laboratories, Hercules, Calif., with permission.)

Citation: Scheppler J, Cassin P, Gambier R. 2000. Protein Studies, p 157-185. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch5
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Citation: Scheppler J, Cassin P, Gambier R. 2000. Protein Studies, p 157-185. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch5
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Figure 19.4

Assembly of the Western blot.

Citation: Scheppler J, Cassin P, Gambier R. 2000. Protein Studies, p 157-185. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch5
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Figure 19.5

Indirect immunodetection of proteins after Western blotting.

Citation: Scheppler J, Cassin P, Gambier R. 2000. Protein Studies, p 157-185. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch5
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References

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1. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248254.
2. Lowry, O. H.,, N. J. Rosebrough,, A. L. Farr,, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265275.
3. Smith, P. K.,, R. I. Krohn,, G. T. Hermanson,, A. K. Mallia,, F. H. Gartner,, M. D. Provenzano,, E. K. Fujimoto,, N. M. Goeke,, B. J. Olson,, and D. C. Klenk. 1985. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150:7685.
4. Walker, J. M. (ed.). 1996. The Protein Protocols Handbook. Humana Press, Totowa, N.J.
5. Alberts, B.,, D. Bray,, J. Lewis,, M. Raff,, K. Roberts,, and J. D. Watson. 1989. Molecular Biology of the Cell, 2nd ed. Garland Publishing, Inc., New York, N.Y.
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7. Ausubel, F. M.,, R. Brent,, R. E. Kingston,, D. D. Moore,, J. G. Seidman,, J. A. Smith,, and K. Struhl (ed.). 1994. Current Protocols in Molecular Biology. Wiley Interscience, New York, N.Y.
8. Belitz, H. D.,, and W. Grosch. 1987. Food Chemistry. Springer-Verlag, Heidelberg, Germany.
9. Brocklehurst, K.,, E. Salih,, R. McKee,, and H. Smith. 1985. Fresh non-fruit latex of Carica papaya contains papain, multiple forms of chymopapain A and papaya proteinase omega. Biochem. J. 228:525527.
10. Kamphuis, I. G.,, K. H. Kalk,, M. B. Swarte,, and J. Drenth. 1984. Structure of papain refined at 1.65 A resolution. J. Mol. Biol. 179:233256.
11. Silva, L. G.,, O. Garcia,, M. T. Lopes,, and C. E. Salas. 1997. Changes in protein profile during coagulation of latex from Carica papaya. Braz. J. Med. Biol. Res. 30:615619.
12. Simpson, B. B.,, and M. C. Ogorzaly. 1995. Economic Botany: Plants in Our World, 2nd ed. McGraw-Hill Book Co., New York, N.Y.
13. Spector, W. S. (ed.). 1956. The Handbook of Biological Data. The W. B. Saunders Co., Philadelphia, Pa.
14. Whitney, E. N.,, C. B. Cataldo,, and S. R. Rolfes. 1998. Understanding Normal and Clinical Nutrition, 5th ed. West/Wadsworth, Belmont, Calif.
15. Worthington, V. (ed.). 1993. Worthington Enzymes and Related Biochemicals Manual. Worthington Biochemical Corporation, Lakewood, NJ.
16. Zpalis, C.,, and A. Beck. 1985. Food Chemistry and Nutritional Biochemistry. John Wiley & Sons, Inc., New York, N.Y.

Tables

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Citation: Scheppler J, Cassin P, Gambier R. 2000. Protein Studies, p 157-185. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch5
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Table 17.1

Bradford assay data: standards and unknown samples

Sample reading minus average reagent blank reading.

Citation: Scheppler J, Cassin P, Gambier R. 2000. Protein Studies, p 157-185. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch5
Generic image for table
Untitled

Citation: Scheppler J, Cassin P, Gambier R. 2000. Protein Studies, p 157-185. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch5
Generic image for table
Untitled

Citation: Scheppler J, Cassin P, Gambier R. 2000. Protein Studies, p 157-185. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch5
Generic image for table
Untitled

Citation: Scheppler J, Cassin P, Gambier R. 2000. Protein Studies, p 157-185. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch5
Generic image for table
Untitled

Citation: Scheppler J, Cassin P, Gambier R. 2000. Protein Studies, p 157-185. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch5
Generic image for table
Untitled

Citation: Scheppler J, Cassin P, Gambier R. 2000. Protein Studies, p 157-185. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch5
Generic image for table
Untitled

Citation: Scheppler J, Cassin P, Gambier R. 2000. Protein Studies, p 157-185. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch5
Generic image for table
Untitled

Citation: Scheppler J, Cassin P, Gambier R. 2000. Protein Studies, p 157-185. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch5
Generic image for table
Untitled

Citation: Scheppler J, Cassin P, Gambier R. 2000. Protein Studies, p 157-185. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch5

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