Gel Electrophoresis

Topics: Gel electrophoresis, SDS-PAGE, Molecular biology Pages: 42 (13826 words) Published: February 28, 2013
Molecular Biology Problem Solver: A Laboratory Guide. Edited by Alan S. Gerstein Copyright © 2001 by Wiley-Liss, Inc. ISBNs: 0-471-37972-7 (Paper); 0-471-22390-5 (Electronic)

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Electrophoresis
Martha L. Booz
Chemical Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . What Is the Safest Approach to Working with Acrylamide? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . What Are the Symptoms of Acrylamide Poisoning? . . . . . . What Is the Medical Response to Accidental Acrylamide Exposure? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . How Can You Dispose of Excess, Unusable Acrylamide? . . What Is the Shelf Life of Acrylamide and Acrylamide Solutions? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Electrical Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . What Are the Requirements for a Safe Work Area? . . . . . . What Are the Requirements for Safe Equipment in Good Working Order? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Polyacrylamide (PAGE) Gels—Before Selecting a Gel: Getting the Best Results for Your Purpose . . . . . . . . . . . . . . . . What Is the Mechanism of Acrylamide Polymerization? . . . What Other Crosslinkers Are Available, and When Should They Be Used? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . How Do You Control Pore Size? . . . . . . . . . . . . . . . . . . . . . . How Do You Calculate %T and %C? . . . . . . . . . . . . . . . . . . . 334 334 335 335 335 336 336 336 337 337 338 338 339 341

I am grateful to Bruce Goodrich for the figure on degassing acrylamide, to Fiona Leung for the data regarding the molecular weight vs. relative mobility curve, and to Lee Olech and Dave Garfin for fruitful discussions about many of the questions in this chapter.

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Why Should You Overlay the Gel? What Should You Use for an Overlay? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regarding Reproducible Polymerization, What Practices Will Ensure That Your Bands Run the Same Way Every Time? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . What Catalyst Concentration Should You Use? . . . . . . . . . . What Is the Importance of Reagent Purity on Protein Electrophoresis and Staining? . . . . . . . . . . . . . . . . . . . . . . . Which Gel Should You Use? SDS-PAGE, Native PAGE or Isoelectric Focusing? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Will Your SDS Gel Accurately Indicate the Molecular Weight of Your Proteins? . . . . . . . . . . . . . . . . . . . . . . . . . . . Should You Use a Straight % Gel or a Gradient Gel? . . . . . What Issues Are Relevant for Isoelectric Focusing? . . . . . . How Can You Resolve Proteins between Approximately 300 and 1000 kDa? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . What Issues Are Critical for Successful Native PAGE? . . . . . . Sample Solubility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Location of Band of Interest . . . . . . . . . . . . . . . . . . . . . . . . . How Can You Be Sure That Your Proteins Have Sufficient Negative Charge to Migrate Well into a Native PAGE Gel? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Buffer Systems for Native PAGE . . . . . . . . . . . . . . . . . . . . . . . What Can Go Wrong with the Performance of a Discontinuous Buffer System? . . . . . . . . . . . . . . . . . . . . . . . . . . What Buffer System Should You Use for Peptide Electrophoresis? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Power Issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Constant Current or Constant Voltage—When and Why? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Why Are Nucleic Acids Almost...
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