Measuring the Success of Pisum sativum leaf cell fractionation: SDS-Polyacrylamide Gel Electrophoresis and Western Blot Analysis
Natalia Rey
080927440
BI338
Lab 1
Measuring the Success of Pisum sativum leaf cell fractionation: SDS-Polyacrylamide Gel Electrophoresis and Western Blot Analysis
Introduction
In order to evaluate the success of cellular fractionation, there are various methods that can be implemented that target specific proteins and/or receptors specific to particular organelles. SDS-PAGE (sodium dedocyl sulfate- polyachrylamide gel electrophoresis) is a technique used to separate proteins based on their mass. As different proteins vary in properties other than mass, the detergent sodium dedocyl sulfate is applied to protein samples (Pokorny, 2012). This disrupts associations by causing the proteins to denature and unfold into a polypeptide rod. It also prevents refolding by coating their surfaces giving them a net negative charge (Becker et al., 2009). The proteins are then transferred to a polyachrylamide gel containing a positively charged anode at the base. The proteins are thus differentiated by mass, causing the smaller proteins to migrate through the gel at a higher rate than larger molecules (Becker et al., 2009). Once the proteins have been separated, a non-specific protein stain is added to the gel to allow for visualization of the proteins. In this case ‘Coomassie blue’ will be used (Pokorny, 2012). To determine specific proteins, Western Blotting was introduced by Towbin et al. in 1979. This technique is now commonly used as it is simple, fast and efficient (Thermo Scientific, 2010). The gel from SDS-PAGE is transferred onto a membrane using and electric field known as electrophoretic transfer. The gel is placed against the membrane eluting the proteins onto the matrix using the force of an electric field (Pokorny, 2012). Once on the membrane, it must be blocked using skim milk and treated with a primary and secondary antibody in order to identify a specific protein. The antibodies that will be used are an anti-Rubisco that will mark the chloroplast
080927440
BI338
Lab 1
Measuring the Success of Pisum sativum leaf cell fractionation: SDS-Polyacrylamide Gel Electrophoresis and Western Blot Analysis
Introduction
In order to evaluate the success of cellular fractionation, there are various methods that can be implemented that target specific proteins and/or receptors specific to particular organelles. SDS-PAGE (sodium dedocyl sulfate- polyachrylamide gel electrophoresis) is a technique used to separate proteins based on their mass. As different proteins vary in properties other than mass, the detergent sodium dedocyl sulfate is applied to protein samples (Pokorny, 2012). This disrupts associations by causing the proteins to denature and unfold into a polypeptide rod. It also prevents refolding by coating their surfaces giving them a net negative charge (Becker et al., 2009). The proteins are then transferred to a polyachrylamide gel containing a positively charged anode at the base. The proteins are thus differentiated by mass, causing the smaller proteins to migrate through the gel at a higher rate than larger molecules (Becker et al., 2009). Once the proteins have been separated, a non-specific protein stain is added to the gel to allow for visualization of the proteins. In this case ‘Coomassie blue’ will be used (Pokorny, 2012). To determine specific proteins, Western Blotting was introduced by Towbin et al. in 1979. This technique is now commonly used as it is simple, fast and efficient (Thermo Scientific, 2010). The gel from SDS-PAGE is transferred onto a membrane using and electric field known as electrophoretic transfer. The gel is placed against the membrane eluting the proteins onto the matrix using the force of an electric field (Pokorny, 2012). Once on the membrane, it must be blocked using skim milk and treated with a primary and secondary antibody in order to identify a specific protein. The antibodies that will be used are an anti-Rubisco that will mark the chloroplast
References: Lodish H, Berk A, Zipursky SL, et al. (2000). Molecular cell biology. (4th ed.). New York: W.H. Freeman. Moore, Claire.(2009) Introduction to Western Blotting. AbD Serotec. Informational Brochure. Overview of Western Blotting. (n.d.).Protein purification, modification and detection: Pierce Protein Research. Retrieved December 2, 2012, from http://www.piercenet.com/browse.cfm?fldID =8259A7B6-7DA6-41CF-9D55-AA6C14F31193 Pokorny, N. (2012). “ Cells- Form & Function Laboratory Manuel. Wilfrid Laurier University, Department of Biology. 19-24. Wilson, Emily. (2010). Lab Report on Western Blot Analyses. Wilfrid Laurier University, Department of Biology.