Molecular Biology Problem Solver: A Laboratory Guide. Edited by Alan S Gerstein opyright◎2001 ISBNS:0-471-37972-7( Paper);0-471 (Electronic) 2 Electrophoresis Martha L booz Chemical Safety What ls the Safest Approach to Working with 334 What Are the Symptoms of Acrylamide Poisoning?.... 335 What Is the Medical Response to Accidental Acrylamide ..335 What ls the Shelf Life of Acrylamide and Acrylamide e?.335 How Can You Dispose of Excess, Unusable Acrylam Solutions? Electrical Safety ..336 What Are the Requirements for Safe Equipment in Good 336 What Are the Requirements for a Safe Work area? Working Order 337 Polyacrylamide(PAGE) Gels--Before Selecting a Gel Getting the best results for Your 337 What Is the Mechanism of Acrylamide Polymerization?.. 338 What Other Crosslinkers are available. and when Should They Be Used? 338 How Do you control pore size How Do You calculate %T and %C? 34 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
331 12 Electrophoresis Martha L. Booz Chemical Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334 What Is the Safest Approach to Working with Acrylamide? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334 What Are the Symptoms of Acrylamide Poisoning? . . . . . . 335 What Is the Medical Response to Accidental Acrylamide Exposure? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335 How Can You Dispose of Excess, Unusable Acrylamide? . . 335 What Is the Shelf Life of Acrylamide and Acrylamide Solutions? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336 Electrical Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336 What Are the Requirements for a Safe Work Area? . . . . . . 336 What Are the Requirements for Safe Equipment in Good Working Order? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337 Polyacrylamide (PAGE) Gels—Before Selecting a Gel: Getting the Best Results for Your Purpose . . . . . . . . . . . . . . . . 337 What Is the Mechanism of Acrylamide Polymerization? . . . 338 What Other Crosslinkers Are Available, and When Should They Be Used? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338 How Do You Control Pore Size? . . . . . . . . . . . . . . . . . . . . . . 339 How Do You Calculate %T and %C? . . . . . . . . . . . . . . . . . . . 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. 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)
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 Is the Importance of Reagent Purity on Protein..343 What Catalyst Concentration Should You Use? Electrophoresis and Staining Which Gel Should You Use? SDS-PaGe, Native PAGe or Will Your SDS Gel Accurately Indicate the Molecular Weight of Your Proteins? 345 Should You Use a Straight Gel or a gradient Gel? 345 What Issues Are Relevant for Isoelectric Focusing? 346 How Can You Resolve Proteins between Al 300 and 1000 kDa? What Issues are Critical for successful native page? 348 348 Location of band of interest 348 How Can You be sure that Your proteins have sufficient N Well into a Native page 348 Buffer Systems for Native PAGE What Can Go Wrong with the Performance of a Discontinuous Buffer Syste What Buffer System Should You Use for Peptide Electrophoresis? 350 Power issues 350 Constant Current or Constant VoltageWhen and Why Are Nucleic Acids Almost Always Separated via Constant Voltage? 352 Why Are Sequencing Gels Electrophoresed under Constant power? 352 Should You Run Two Sequencing Cells off the Same Power Supply under Constant Power Improving Resolution and Clarity of Protein Gels 353 How Can You Generate Reproducible Gels with Perfect Bands Every Time? Sample Preparation--Problems with Protein Samples 353 What Procedures and Strategies Should Be Used to Optimize Protein Sample Preparation Is the Problem Caused by Sample Preparation or by the Electrophoresis? 332 Booz
Why Should You Overlay the Gel? What Should You Use for an Overlay? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341 Regarding Reproducible Polymerization, What Practices Will Ensure That Your Bands Run the Same Way Every Time? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341 What Catalyst Concentration Should You Use? . . . . . . . . . . 343 What Is the Importance of Reagent Purity on Protein Electrophoresis and Staining? . . . . . . . . . . . . . . . . . . . . . . . 343 Which Gel Should You Use? SDS-PAGE, Native PAGE or Isoelectric Focusing? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345 Will Your SDS Gel Accurately Indicate the Molecular Weight of Your Proteins? . . . . . . . . . . . . . . . . . . . . . . . . . . . 345 Should You Use a Straight % Gel or a Gradient Gel? . . . . . 345 What Issues Are Relevant for Isoelectric Focusing? . . . . . . 346 How Can You Resolve Proteins between Approximately 300 and 1000kDa? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347 What Issues Are Critical for Successful Native PAGE? . . . . . . 348 Sample Solubility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348 Location of Band of Interest . . . . . . . . . . . . . . . . . . . . . . . . . 348 How Can You Be Sure That Your Proteins Have Sufficient Negative Charge to Migrate Well into a Native PAGE Gel? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348 Buffer Systems for Native PAGE . . . . . . . . . . . . . . . . . . . . . . . 349 What Can Go Wrong with the Performance of a Discontinuous Buffer System? . . . . . . . . . . . . . . . . . . . . . . . . . . 349 What Buffer System Should You Use for Peptide Electrophoresis? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 350 Power Issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 350 Constant Current or Constant Voltage—When and Why? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351 Why Are Nucleic Acids Almost Always Separated via Constant Voltage? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352 Why Are Sequencing Gels Electrophoresed under Constant Power? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352 Should You Run Two Sequencing Cells off the Same Power Supply under Constant Power? . . . . . . . . . . . . . . . . . . . . . 352 Improving Resolution and Clarity of Protein Gels . . . . . . . . . 353 How Can You Generate Reproducible Gels with Perfect Bands Every Time? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353 Sample Preparation—Problems with Protein Samples . . . . . . 353 What Procedures and Strategies Should Be Used to Optimize Protein Sample Preparation? . . . . . . . . . . . . . . . 353 Is the Problem Caused by Sample Preparation or by the Electrophoresis? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354 332 Booz
Is the Problem Caused by the Sample or the Sample Buffer? 354 How Do You Choose a Detergent for ieF or Native 354 What Other Additives Can be used to enhance protein Agarose Electrophoresis ..355 What Is agarose What Is Electroendosmosis PPPppriate for Sizing M or EEO)? 355 Are double-Stranded marke Large Single-Stranded(Not Oligonucleotide) DNA? 356 What Causes Nucleic Acids to Migrate at Unexpected 356 What Causes Commercial Preparations of Nucleic Acid What Causes Fuzzy Bands ..357 Elution of nucleic acids and proteins from gels 357 Detection 357 What Should You Consider before Selecting a Stain, ::357 Will the choice of stain Affect a downstream Application Is Special Equipment Needed to View the Stain? 36 How Much Time Is Required for the Various What If You Need to Quantify Your Stained Protein? What Causes High Background Staining? 362 Will the Presence of stain on Western-Blotted Proteins Interfere with Subsequent Hybridization or Antibody Detection Reactions? 363 Does Ethidium Bromide Interfere with the common Enzymatic Manipulation of Nucleic Acids 363 Standardizing Your Gels 363 What Factors Should Be Considered before Selecting a Molecular Weight Marker? 363 Are Double-Stranded Markers Appropriate for Sizing Large(Not Oligonucleotide) Single-Stranded DNA? If Not, Which Markers Are Recommended 364 Can a Pre-stained Standard Be Applied to Determine the Molecular Weight of an Unknown Protein? 364 How Do You Determine Molecular Weight on a Western blot? 365 What Are the Options for Determining pl and Molecular Weight on a 2-D Gel? 365 333
Is the Problem Caused by the Sample or the Sample Buffer? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354 How Do You Choose a Detergent for IEF or Native PAGE? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354 What Other Additives Can Be Used to Enhance Protein Solubility? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355 Agarose Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355 What Is Agarose? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355 What Is Electroendosmosis (-Mr or EEO)? . . . . . . . . . . . . . . 355 Are Double-Stranded Markers Appropriate for Sizing Large Single-Stranded (Not Oligonucleotide) DNA? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 356 What Causes Nucleic Acids to Migrate at Unexpected Migration Rates? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 356 What Causes Commercial Preparations of Nucleic Acid Markers to Smear? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 356 What Causes Fuzzy Bands? . . . . . . . . . . . . . . . . . . . . . . . . . . . 357 Elution of Nucleic Acids and Proteins from Gels . . . . . . . . . . . 357 Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357 What Should You Consider before Selecting a Stain? . . . . . . 357 Will the Choice of Stain Affect a Downstream Application? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359 Is Special Equipment Needed to View the Stain? . . . . . . . . . 361 How Much Time Is Required for the Various Stains? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361 What If You Need to Quantify Your Stained Protein? . . . . . 361 What Causes High Background Staining? . . . . . . . . . . . . . . . 362 Will the Presence of Stain on Western-Blotted Proteins Interfere with Subsequent Hybridization or Antibody Detection Reactions? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363 Does Ethidium Bromide Interfere with the Common Enzymatic Manipulation of Nucleic Acids? . . . . . . . . . . . . . 363 Standardizing Your Gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363 What Factors Should Be Considered before Selecting a Molecular Weight Marker? . . . . . . . . . . . . . . . . . . . . . . . . . . 363 Are Double-Stranded Markers Appropriate for Sizing Large (Not Oligonucleotide) Single-Stranded DNA? If Not, Which Markers Are Recommended? . . . . . . . . . . . . 364 Can a Pre-stained Standard Be Applied to Determine the Molecular Weight of an Unknown Protein? . . . . . . . . . . . 364 How Do You Determine Molecular Weight on a Western Blot? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365 What Are the Options for Determining pI and Molecular Weight on a 2-D Gel? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365 Electrophoresis 333
How Do You Measure the ph Gradient of a Tube lEF Gel or an 366 Troubleshooting∴. 368 What ls This Band Going All the Way across a Silver- Stained Gel, between Approximately 55 and 65kDa How Can You Stop the Buffer Leaking from the Upper Chamber of a vertical slab cell Bibliography 368 Appendix A: Procedure for Degassing Acrylamide Gel 37 Dangerously high voltage and acrylamide, a neurotoxin and sus- pected carcinogen, are inescapable elements of electrophoresis. Proper personal protection and good laboratory practice will min imize the risk of harming yourself or your colleagues. CHEMICAL SAFETY What Is the Safest Approach to Working with Acrylamide? Unpolymerized, monomeric acrylamide is a neurotoxin in any form. Bis-acrylamide is equally dangerous. Protect yourself by wearing gloves, a lab coat, and safety glasses, and never pipet acrylamide solutions by mouth Acrylamide powders should be weighed and solutions prepared in a ventilated hood Acrylamide can be detected in the air above a beaker of acrylamide solution and throughout the laboratory Values in the single-digit ppm range are detected above a 10% solution at room temperature(Figure 12. 1).The detection method involves r samples through an acrylamide- binding column, and analyzing the eluant via HPLC (Dow Chemical Company, 1988). The MSDS for acrylamide gives the OSHa per- missible exposure limit for acrylamide as 0.3mg/m' for personal exposure in an industrial setting The use of pre-cast gels and pre-mixed acrylamide solutions can reduce exposure to acrylamide and bis-acrylamide. Even after polymerization, a small fraction of the acrylamide remains in the neurotoxic monomeric form. Wear gloves when handling a poly merized gel. If you need to cast your own gels, we suggest you use pre-mixed acrylamide solutions, which are also available from many vendors. The pre-mixed solutions avoid the weighing and mixing steps, and generally have a long storage life 334 Booz
How Do You Measure the pH Gradient of a Tube IEF Gel or an IPG Gel? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 368 What Is This Band Going All the Way across a SilverStained Gel, between Approximately 55 and 65kDa? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 368 How Can You Stop the Buffer Leaking from the Upper Chamber of a Vertical Slab Cell? . . . . . . . . . . . . . . . . . . . . . 368 Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 368 Appendix A: Procedure for Degassing Acrylamide Gel Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371 Dangerously high voltage and acrylamide, a neurotoxin and suspected carcinogen, are inescapable elements of electrophoresis. Proper personal protection and good laboratory practice will minimize the risk of harming yourself or your colleagues. CHEMICAL SAFETY What Is the Safest Approach to Working with Acrylamide? Unpolymerized, monomeric acrylamide is a neurotoxin in any form. Bis-acrylamide is equally dangerous. Protect yourself by wearing gloves, a lab coat, and safety glasses, and never pipet acrylamide solutions by mouth. Acrylamide powders should be weighed and solutions prepared in a ventilated hood. Acrylamide can be detected in the air above a beaker of acrylamide solution and throughout the laboratory. Values in the single-digit ppm range are detected above a 10% solution at room temperature (Figure 12.1).The detection method involves passing air samples through an acrylamide-binding column, and analyzing the eluant via HPLC (Dow Chemical Company, 1988). The MSDS for acrylamide gives the OSHA permissible exposure limit for acrylamide as 0.3mg/m3 for personal exposure in an industrial setting. The use of pre-cast gels and pre-mixed acrylamide solutions can reduce exposure to acrylamide and bis-acrylamide. Even after polymerization, a small fraction of the acrylamide remains in the neurotoxic monomeric form. Wear gloves when handling a polymerized gel. If you need to cast your own gels, we suggest you use pre-mixed acrylamide solutions, which are also available from many vendors. The pre-mixed solutions avoid the weighing and mixing steps, and generally have a long storage life. 334 Booz
Figure 12.1 Vapor phase concentrations of acry. lamide-water solutions (10- 50% Industries Inc., 1995. Re- 35% 8 15% What Are the Symptoms of Acrylamide poisoning? The initial symptoms of acrylamide poisoning on the skin are peeling of the skin at the point of contact, followed by tingling and numbness in the d ans(touch ingestion, inhalation) continues, muscular weakness, difficulty maintaining balance, and clumsiness in walking and in the use of the hands may develop. A large, acute exposure can produce con fusion, disorientation, slurred speech and ataxia(severe loss of balance). Muscular weakness and numbness in the extremities may also follow. Anyone exposed to any form of acrylamide should be immediately examined by a medical doctor(Bio-Rad Laboratories, MSDS, 2000) What Is the Medical Response to Accidental Acrylamide Exposure? On your skin: Wash the affected skin several times with soap for at least 15 minutes under running water In your mouth: Rinse your mouth immediately with water and seek medical attention immediately Swallowed or inhaled: If swallowed, do not induce vomiting attention immediately. If breathed fresh air, and seek medical attention immediately(Bio-Rad Laboratories, MSDS, 2000) How Can You Dispose of Excess, Unusable Acrylamide? Check with your institutional or local county environmental area. The safest Electrophoresis
What Are the Symptoms of Acrylamide Poisoning? The initial symptoms of acrylamide poisoning on the skin are peeling of the skin at the point of contact, followed by tingling and numbness in the exposed area. If exposure by any means (touch, ingestion, inhalation) continues, muscular weakness, difficulty maintaining balance, and clumsiness in walking and in the use of the hands may develop. A large, acute exposure can produce confusion, disorientation, slurred speech and ataxia (severe loss of balance). Muscular weakness and numbness in the extremities may also follow. Anyone exposed to any form of acrylamide should be immediately examined by a medical doctor (Bio-Rad Laboratories, MSDS, 2000). What Is the Medical Response to Accidental Acrylamide Exposure? On your skin: Wash the affected skin several times with soap for at least 15 minutes under running water. In your mouth: Rinse your mouth immediately with water and seek medical attention immediately. Swallowed or inhaled: If swallowed, do not induce vomiting. Seek medical attention immediately. If breathed in, get to fresh air, and seek medical attention immediately (Bio-Rad Laboratories, MSDS, 2000). How Can You Dispose of Excess, Unusable Acrylamide? Check with your institutional or local county environmental regulators for the disposal requirements in your area. The safest Electrophoresis 335 10 20 30 40 50 60 70 80 90 100 Temperature °C 10% 50% 25% 35% 15% 1000 100 10 1 Vapor Phase Concentration, ppm Figure 12.1 Vapor phase concentrations of acrylamide-water solutions (10– 50% acrylamide). Cytec Industries Inc., 1995. Reprinted by permission of Cytec Inc