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
N, N'-methylenebisacrylamide crosslink Initiator and Catalyst (NH4hS,OS/TEME NH2 NH Figure 12.2 Polymerization of acrylamide. Reproduced with permission from Bio-Rad Laboratories How Do You control pore size? Pore size is most efficiently and predictably regulated by manip- ulating the concentration of acrylamide in the gel. Pore size will ge with the amount of crosslinker but the effect is minimal crosslinker usually present in gels(2.7-5%) Practical experience with various ratios of acrylamide bis shown that it is best to change pore size by changing the 339
How Do You Control Pore Size? Pore size is most efficiently and predictably regulated by manipulating the concentration of acrylamide in the gel. Pore size will change with the amount of crosslinker, but the effect is minimal and less predictable (Figure 12.3). Note the greater impact of acrylamide concentration on pore size, especially at the levels of crosslinker usually present in gels (2.7–5%). Practical experience with various ratios of acrylamide:bis have shown that it is best to change pore size by changing the acryElectrophoresis 339 Figure 12.2 Polymerization of acrylamide. Reproduced with permission from Bio-Rad Laboratories
10/20 10/10 107 2.3/5 20/5 40/5 10/1 Figure 12.3 Electron micrograph of polyacrylamide gels of various %T, showing the hange in pore size with the change in %Tand %C From Ruechel, Steere, and Erbe(1978, Fig 3, P. 569). Reprinted from Journal of Chromatography, volume 166, Ruechel, R, Steere, R, and Erbe, E Transmission-electron Microscopic Observations of Freeze-etched Poly acrylamide gels. pp 563-575. 1978. With permission from Elsevier Science 340 Booz
340 Booz 10/20 10/10 10/7 10/6 10/2 10/1 10/0.2 10/5 2.3/5 5/5 20/5 40/5 Figure 12.3 Electron micrograph of polyacrylamide gels of various %T, showing the change in pore size with the change in %T and %C. From Rüechel, Steere, and Erbe (1978, Fig. 3, p. 569). Reprinted from Journal of Chromatography, volume 166, Ruechel, R., Steere, R., and Erbe, E. Transmission-electron Microscopic Observations of Freeze-etched Polyacrylamide gels. pp. 563–575. 1978. With permission from Elsevier Science