【资源】Electrophoresis 知识
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Electrophoresis其实并不简单
我们要学的还很多
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
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
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
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 Silver-
Stained 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
我们要学的还很多
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
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
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
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 Silver-
Stained 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
