2010 Bio 311 Lab E Manual (part 2)

2010 Bio 311 Lab E Manual (part 2) - E2. SDS-PAGE Lab...

Info iconThis preview shows pages 1–2. Sign up to view the full content.

View Full Document Right Arrow Icon
133 E2. . Lab Overview: The partially pure EGF protein from the Protein Purification lab will be viewed by SDS Polyacrylamide gel electrophoresis (SDS PAGE). All the proteins on this gel will be blotted onto a PVDF membrane. You will probe and detect EGFP on the membrane by Western Blotting next week. Background Proteins are usually positively or negatively charged based on the amino acids present in them. For example, a basic protein will tend to have a lot of lysines and arginines, while an acidic protein will have more glutamic acids and aspartic acids. The overall charge of a protein will influence its migration during electrophoresis and approximating the molecular weight of the protein will be difficult. In order to look at proteins based on size , the strongly anionic detergent SDS is added. SDS is a detergent commonly found in shampoo. SDS has a charged end and a hydrophobic end. It denatures and coats proteins with negative charges. In conjunction with a reducing agent, dithiothreitol (DTT) that breaks disulfide bonds, the proteins become negatively charged, extended rods. Since the contribution of charge by the SDS is greater than that of the protein itself, all proteins treated with SDS have a net negative charge, and will migrate in an electric field toward the positive electrode. Proteins will also have the same charge to mass ratio, such that they migrate based on size. If these proteins are electrophoresed in a polyacrylamide gel with SDS, they will thus migrate according to size. Polyacrylamide is a gel matrix made of acrylamide monomers that are linked together with bis- acrylamide. This polymerization reaction is catalyzed by ammonium persulfate and TEMED. Because polyacrylamide has pores of varying sizes depending on the concentration used, it acts as a molecular sieve like agarose. Large proteins (100 kD) travel slower and tend to be found towards the top of the gel, while small proteins (20 kD) migrate faster and tend to be found near the bottom of the gel. The pHs of the buffers are important to this gel system. The top portion of the gel is made with a buffer that has a pH of 6.8. The lower portion is made with a buffer that has a pH of 8.8. At acidic pH (6.8), the glycine in the running buffer is uncharged and backs up in the gel, preventing the proteins from advancing. The proteins become "stacked" up again the uncharged glycine. Once the glycine and proteins hit the basic pH, the glycine becomes charged and rapidly moves toward the positive electrode. The proteins separate based on molecular weight. You will be adding marker that contains colorized proteins of various sizes for visual aid. In order to visualize proteins after PAGE, you must stain them. Coomassie blue R250 is a textile dye used to stain proteins. Use caution since it will stain your hands, as well as anything that comes into contact with it. Coomassie blue stains all proteins indiscriminately therefore you cannot use it to detect a specific protein.
Background image of page 1

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Image of page 2
This is the end of the preview. Sign up to access the rest of the document.

This note was uploaded on 03/28/2011 for the course BIO 311 taught by Professor Staff during the Fall '08 term at SUNY Stony Brook.

Page1 / 11

2010 Bio 311 Lab E Manual (part 2) - E2. SDS-PAGE Lab...

This preview shows document pages 1 - 2. Sign up to view the full document.

View Full Document Right Arrow Icon
Ask a homework question - tutors are online