武汉大学生命科学学院：《分子生物学》（英文版）12 Electrophoresis.pdf_大学文库

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 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 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

way to dispose of a small amount of liquid acrylamide is to polymerize it in the hood in a closed plastic bag set into a beaker surrounded by a very large, tightly fastened plastic bag, to prevent attering as the acrylamide polymerizes If you have more than 100ml to dispose of, contact your local environmental safety officers to determine your recommended procedure. Acrylamide solutions emit significant heat during polymerization, and polymerization of large volumes of acryla mide can be explosive due to rapid heat buildup( dow Chemical Company, 1988; Cytec Industries, 1995; Bio-Rad Laboratories, 2000) Acrylamide and bis-acrylamide powders must be disposed of as solid hazardous waste. Consult your local environmental safety What Is the Shelf Life of Acrylamide and Acrylamide Solutions? Commercially prepared acrylamide solutions are stable for as g as one year, unopened, and for six months after opening. The igh purity of the solution components and careful monitoring throughout the manufacturing process provides extended shelf life. The lifetime of homemade solutions similarly depends on the purity of the acrylamide and bis-acrylamide, the cleanliness of the laboratory dishes, and the purity of the water used to make he solutions a Solid acrylamide breaks down with time due to oxidation and TV light, producing acrylic acid and ammonia. Acrylic acid in a gel can cause fuzzy bands, or fuzzy spots in the case of 2-D gels, streaking and smearing, and poor resolution(Allen and Budowle 1994) Acrylamide decomposition occurs more quickly in solution, and it can be accelerated by any impurities within the water(Allen and Budowle, 1994). Thus acrylamide powder should be stored airtight at room temperature, and acrylamide solutions should be stored at 4C. both in the dark Production facilities must establish standards and measures to determine the effective lifetime of unpolymerized acrylamide ELECTRICAL SAFETY What Are the Requirements for a safe Work Area? The voltages used in electrophoresis can be dangerous, fires have occurred due to problems with electrophoresis cell 336 Booz

way to dispose of a small amount of liquid acrylamide is to polymerize it in the hood in a closed plastic bag set into a beaker surrounded by a very large, tightly fastened plastic bag, to prevent spattering as the acrylamide polymerizes. If you have more than 100ml to dispose of, contact your local environmental safety officers to determine your recommended procedure. Acrylamide solutions emit significant heat during polymerization, and polymerization of large volumes of acrylamide can be explosive due to rapid heat buildup (Dow Chemical Company, 1988; Cytec Industries, 1995; Bio-Rad Laboratories, 2000). Acrylamide and bis-acrylamide powders must be disposed of as solid hazardous waste. Consult your local environmental safety office. What Is the Shelf Life of Acrylamide and Acrylamide Solutions? Commercially prepared acrylamide solutions are stable for as long as one year, unopened, and for six months after opening. The high purity of the solution components and careful monitoring throughout the manufacturing process provides extended shelf life. The lifetime of homemade solutions similarly depends on the purity of the acrylamide and bis-acrylamide, the cleanliness of the laboratory dishes, and the purity of the water used to make the solutions. Solid acrylamide breaks down with time due to oxidation and UV light, producing acrylic acid and ammonia. Acrylic acid in a gel can cause fuzzy bands, or fuzzy spots in the case of 2-D gels, streaking and smearing, and poor resolution (Allen and Budowle, 1994).Acrylamide decomposition occurs more quickly in solution, and it can be accelerated by any impurities within the water (Allen and Budowle, 1994). Thus acrylamide powder should be stored airtight at room temperature, and acrylamide solutions should be stored at 4°C, both in the dark. Production facilities must establish standards and measures to determine the effective lifetime of unpolymerized acrylamide solutions. ELECTRICAL SAFETY What Are the Requirements for a Safe Work Area? The voltages used in electrophoresis can be dangerous, and fires have occurred due to problems with electrophoresis cells.The 336 Booz

following precautions should be observed to prevent accidents nd fires There should be no puddles of liquid on the horizontal surfaces of the electrophoresis cell The area around the power supply and cell should be dry The area for at least 6 inches around the power supply and cell should be bare of clutter and other equipment. Clear space means any fire or accident can be more easily controlled What Are the Requirements for Safe Equipment in Good Working Order? The wires connecting the cell to the power supply must be in good condition, not worn or cracked, and the banana plugs and jacks must be in good condition, not corroded or worn. Broken or worn wires can cause rapid changes in resistance, slow elec trophoresis or a halt in the run. All cables and connectors must be inspected regularly for breaks and wear The banana plugs on the ends of the wires should be rer rom the power supply at the end of the run by pulling them straight out. Grasp the plug, not the wire. If pulled at an angle, the solder joint attaching the banana plugs to the wires can loosen and cause the loss of the electrical circuit. on the cell core electrode banana posts with flattened baskets do not make good contact with the banana jack in the cell lid, and should be replaced. The banana jacks(female part) in the cell lid should be inspected regularly to make sure there is no corrosion Before starting an electrophoresis run, dry any liquid on the horizontal surfaces of the cell, especially near the banana plugs and jacks. Any liquid on the horizontal surfaces of the cell can arc during the run, damaging the cell and stopping the electrophoresis. POLYACRYLAMIDE (PAGE GELS-BEFORE SELECTING A GEL: GETTING THE BEST RESULTS FOR YOUR PURPOSE Before choosing which gel to use, it is important to consider several questions, all of which can help you choose the gel will give you the best results for your purpose. The next graphs provide information on how to select a gel percentage or pore size, when to use SDS-PAGE and when native PAGE, what buffer system to use, which crosslinker to use, and degree of resolution needed 337

following precautions should be observed to prevent accidents and fires. • There should be no puddles of liquid on the horizontal surfaces of the electrophoresis cell. • The area around the power supply and cell should be dry. • The area for at least 6 inches around the power supply and cell should be bare of clutter and other equipment. Clear space means any fire or accident can be more easily controlled. What Are the Requirements for Safe Equipment in Good Working Order? The wires connecting the cell to the power supply must be in good condition, not worn or cracked, and the banana plugs and jacks must be in good condition, not corroded or worn. Broken or worn wires can cause rapid changes in resistance, slow electrophoresis or a halt in the run. All cables and connectors must be inspected regularly for breaks and wear. The banana plugs on the ends of the wires should be removed from the power supply at the end of the run by pulling them straight out. Grasp the plug, not the wire. If pulled at an angle, the solder joint attaching the banana plugs to the wires can loosen and cause the loss of the electrical circuit. On the cell core, electrode banana posts with flattened baskets do not make good contact with the banana jack in the cell lid, and should be replaced. The banana jacks (female part) in the cell lid should be inspected regularly to make sure there is no corrosion. Before starting an electrophoresis run, dry any liquid on the horizontal surfaces of the cell, especially near the banana plugs and jacks. Any liquid on the horizontal surfaces of the cell can arc during the run, damaging the cell and stopping the electrophoresis. POLYACRYLAMIDE (PAGE) GELS—BEFORE SELECTING A GEL: GETTING THE BEST RESULTS FOR YOUR PURPOSE Before choosing which gel to use, it is important to consider several questions, all of which can help you choose the gel that will give you the best results for your purpose. The next paragraphs provide information on how to select a gel percentage or pore size, when to use SDS-PAGE and when native PAGE, what buffer system to use, which crosslinker to use, and degree of resolution needed. Electrophoresis 337

What Is the Mechanism of Acrylamide Polymerization Most protocols use acrylamide and the crosslinker bis- acrylamide(bis)for the gel matrix. TEMED(N, N, N, N-tetram- ethylethylenediamine)and ammonium persulfate are used to cat alyze the polymerization of the acrylamide and bis TEMED,a base, interacts with ammonium persulfate at neutral to basic pl to produce free radicals. The free radical form of ammonium per sulfate initiates the polymerization reaction via the addition of a vinyl group(Figure 12.2). At an acidic pH, other catalysts must be used,as described in Andrews(1986), Hames and Rickwood (1981), and Caglio and Righetti (1993) What Other Crosslinkers Are Available. and when should They Be Used? Bis-acrylamide is the only crosslinker in common use today. There are others available, for specialty applications. DhEBa (N, N-dihydroxyethylene-bis-acrylamide) and DATD (N,N diallyltartardiamide) were both used historically with tube gels and radioactive samples(before slab gels came into common use) The tube gels were cut into thin discs, the disks were dissolved with periodic acid, and the radioactivity in the disks was counted in a scintillation counter. Of course the periodic acid destroyed some amino acids, so these crosslinkers are not useful for edman sequencing or mass spectrometry Another crosslinker, BAC (bis-acrylylcystamine) can be dis- solved by beta-mercaptoethanol. It is useful for nucleic acid lectrophoresis (Hansen, 1981). However, proteins containing disulfide bonds do not separate on a BAC gel. The subunits with he sulfhydryl moiety bind to the gel matrix close to the origin of he gel, and separation does not occur, So BAC is not recom mended for preparative protein electrophoresis, though it is useful for proteins which do not contain any sulfhydryl bonds. One other crosslinker, piperazine diacrylamide(pda),can replace bis-acrylamide in isoelectric focusing(classical tube gel or flatbed gel) experiments. PDA imparts greater mechanical strength to a polyacrylamide gel, and this is desired at the low acrylamide concentrations used in isoelectric focusing(IEF gels) Some proteomics researchers use PDa to crosslink the 2nddimen sion SDs-PAge slab gels as well, because of the increased stained gel is much better when PDA is used(Hochstrasser, 192 mechanical strength, and because the background of For further information on these crosslinkers see allen and Budowle. 1994 338 Booz

What Is the Mechanism of Acrylamide Polymerization? Most protocols use acrylamide and the crosslinker bisacrylamide (bis) for the gel matrix. TEMED (N,N,N¢,N¢-tetramethylethylenediamine) and ammonium persulfate are used to catalyze the polymerization of the acrylamide and bis. TEMED, a base, interacts with ammonium persulfate at neutral to basic pH to produce free radicals. The free radical form of ammonium persulfate initiates the polymerization reaction via the addition of a vinyl group (Figure 12.2). At an acidic pH, other catalysts must be used, as described in Andrews (1986), Hames and Rickwood (1981), and Caglio and Righetti (1993). What Other Crosslinkers Are Available, and When Should They Be Used? Bis-acrylamide is the only crosslinker in common use today. There are others available, for specialty applications. DHEBA (N,N¢-dihydroxyethylene-bis-acrylamide) and DATD (N,N¢- diallyltartardiamide) were both used historically with tube gels and radioactive samples (before slab gels came into common use). The tube gels were cut into thin discs, the disks were dissolved with periodic acid, and the radioactivity in the disks was counted in a scintillation counter. Of course the periodic acid destroyed some amino acids, so these crosslinkers are not useful for Edman sequencing or mass spectrometry. Another crosslinker, BAC (bis-acrylylcystamine) can be dissolved by beta-mercaptoethanol. It is useful for nucleic acid electrophoresis (Hansen, 1981). However, proteins containing disulfide bonds do not separate on a BAC gel. The subunits with the sulfhydryl moiety bind to the gel matrix close to the origin of the gel, and separation does not occur, so BAC is not recommended for preparative protein electrophoresis, though it is useful for proteins which do not contain any sulfhydryl bonds. One other crosslinker, piperazine diacrylamide (PDA), can replace bis-acrylamide in isoelectric focusing (classical tube gel or flatbed gel) experiments. PDA imparts greater mechanical strength to a polyacrylamide gel, and this is desired at the low acrylamide concentrations used in isoelectric focusing (IEF gels). Some proteomics researchers use PDA to crosslink the 2nd dimension SDS-PAGE slab gels as well, because of the increased mechanical strength, and because the background of a silver stained gel is much better when PDA is used (Hochstrasser, 1988). For further information on these crosslinkers, see Allen and Budowle, 1994. 338 Booz