lamide concentration. A 19: 1 ratio of acrylamide to bis(5%C see below for calculation of C)is used in low concentration gels, such as IEF gels, and sequencing gels, to impart greater mechani cal strength to the gel. A 29: 1 ratio(3. 4%C)is used for concen trations of acrylamide from 8% to 12%, and a 37. 5: 1 ratio(2.67% C) is used for concentrations of acrylamide above 12% to provide flexibility to the gel. SDS-PAGE and native gels are usually run at 10% to 12%. For comparison, a 12% acrylamide gel with a 5% crosslinker concentration will be brittle and will tear easily How Do You calculate %T and %C? Percent T is %T=(g acrylamide +g bis-acrylamide)/100ml water×100 Percent C is %C=(g bis-acrylamide) /(g acrylamide +g bis acrylamide)×100 Note that %C is not the grams bis-acrylamide/100ml water, but rather the percentage of crosslinker as a function of the total weight of acrylamide and bis-acrylamide used Why should You Overlay the Gel? What Should You Use fo r an An overlay is essential for adequate resolution. If you do not overlay, the bands will have the shape of a meniscus. Two closely spaced bands will overlap; the middle of the top band will extend down between the front and back of the bottom band Overlay the gel during polymerization will prevent this problem Common overlays are best quality water, the buffer used in the gel at a lx dilution, and water-saturated t-butanol. The choice is a matter of personal preference. Many researchers prefer the alcohol overlay because it will not mix with the gel solution lle, an, alcohol will turn acrylic plastic(Perspex) from clear to white, and it is difficult to pipet without spills Regarding Reproducible Polymerization, What Practices Will Ensure That Your Bands Run the Same Way Every Time? Reproducible polymerization is one of the most important ways to ensure that your samples migrate as sharp, thin bands to the same location in the gel every time. Attention to polymerization will also help keep the background of your stained gels low. Acry lamide polymerization is affected by the amount of oxygen gas dissolved in the solution the concentrations and condition of the Electrophoresis 34
lamide concentration. A 19 :1 ratio of acrylamide to bis (5% C; see below for calculation of C) is used in low concentration gels, such as IEF gels, and sequencing gels, to impart greater mechanical strength to the gel. A 29 :1 ratio (3.4% C) is used for concentrations of acrylamide from 8% to 12%, and a 37.5 : 1 ratio (2.67% C) is used for concentrations of acrylamide above 12% to provide flexibility to the gel. SDS-PAGE and native gels are usually run at 10% to 12%. For comparison, a 12% acrylamide gel with a 5% crosslinker concentration will be brittle and will tear easily. How Do You Calculate %T and %C? Percent T is %T = (g acrylamide + g bis-acrylamide)/100 ml water ¥ 100. Percent C is %C = (g bis-acrylamide)/(g acrylamide + g bisacrylamide) ¥ 100. Note that %C is not the grams bis-acrylamide/100 ml water, but rather the percentage of crosslinker as a function of the total weight of acrylamide and bis-acrylamide used. Why Should You Overlay the Gel? What Should You Use for an Overlay? An overlay is essential for adequate resolution. If you do not overlay, the bands will have the shape of a meniscus. Two closely spaced bands will overlap; the middle of the top band will extend down between the front and back of the bottom band. Overlaying the gel during polymerization will prevent this problem. Common overlays are best quality water, the buffer used in the gel at a 1¥ dilution, and water-saturated t-butanol. The choice is a matter of personal preference. Many researchers prefer the alcohol overlay because it will not mix with the gel solution. However, alcohol will turn acrylic plastic (Perspex) from clear to white, and it is difficult to pipet without spills. Regarding Reproducible Polymerization,What Practices Will Ensure That Your Bands Run the Same Way Every Time? Reproducible polymerization is one of the most important ways to ensure that your samples migrate as sharp, thin bands to the same location in the gel every time. Attention to polymerization will also help keep the background of your stained gels low. Acrylamide polymerization is affected by the amount of oxygen gas dissolved in the solution, the concentrations and condition of the Electrophoresis 341
catalysts, the temperatures and ph of the stock solutions, and the purity of the gel components. The following paragraphs discuss how to ensure reproducible polymerization and therefore repro- ducible, excellent gels Eliminate Dissolved Oxygen Oxygen quenches the free radicals generated by TEMED and APS, thus inhibiting the polymerization reaction. Dissolved oxygen must be eliminated via degassing with a bench vacuum or better(20-23 inches of mercury or better) for at least 15 to 30 minutes with stirring(see Appendix A). To achieve reproducible polymerization and consistent pore size, allow the gel solutions, thich should be stored in the cold to inhibit breakdown to come to room temperature before casting a gel. Note that cold gel solu tions contain more dissolved oxygen, and low temperature directly inhibits the polymerization reaction. If the temperature during polymerization is not controlled, the pore size will vary from day to day. Symptoms of problems with Catalyst Potency The best indicator of a problem catalyst is poor polymeriza- tion of the gel. If you're confident that you have good quality chemicals and water, and have degassed your solutions t remove oxygen, and still the sides of the wells do not polymerize around the teeth of the comb, a degraded catalyst is the likely Separation of the gel from the spacers also indicates poor poly- merization;the dye front will migrate in the shape of a frown. A third symptom of poor polymerization is schlieren in the body of he gel. Schlieren are swirls, changes in the refractive index of the gel, where polymerization has been very slow or has not occurred The gel has no structure at the location of the schlieren. It breaks apart in pieces at the schlieren lines, when removed from the cas- sette Schlieren can also be caused by inadequate mixing of the gel olution before pouring it into the gel cassette It is difficult to predict the potency of TEMED unless you know its history of use. TEMED is very hygroscopic and will degrade within six months of purchase if it becomes contaminated with water. Therefore store TEMED in a desiccator at room tempera- ture if you use it frequently, or at 4C if you use it less than once a week. Cold TEMED must be warmed to room temperature before the bottle is opened to prevent condensation from con- taminating the TEMED liquid 342 Booz
catalysts, the temperatures and pH of the stock solutions, and the purity of the gel components. The following paragraphs discuss how to ensure reproducible polymerization and therefore reproducible, excellent gels. Eliminate Dissolved Oxygen Oxygen quenches the free radicals generated by TEMED and APS, thus inhibiting the polymerization reaction. Dissolved oxygen must be eliminated via degassing with a bench vacuum or better (20–23 inches of mercury or better) for at least 15 to 30 minutes with stirring (see Appendix A). To achieve reproducible polymerization and consistent pore size, allow the gel solutions, which should be stored in the cold to inhibit breakdown, to come to room temperature before casting a gel. Note that cold gel solutions contain more dissolved oxygen, and low temperature directly inhibits the polymerization reaction. If the temperature during polymerization is not controlled, the pore size will vary from day to day. Symptoms of Problems with Catalyst Potency The best indicator of a problem catalyst is poor polymerization of the gel. If you’re confident that you have good quality chemicals and water, and have degassed your solutions to remove oxygen, and still the sides of the wells do not polymerize around the teeth of the comb, a degraded catalyst is the likely explanation. Separation of the gel from the spacers also indicates poor polymerization; the dye front will migrate in the shape of a frown. A third symptom of poor polymerization is schlieren in the body of the gel. Schlieren are swirls, changes in the refractive index of the gel, where polymerization has been very slow or has not occurred. The gel has no structure at the location of the schlieren. It breaks apart in pieces at the schlieren lines, when removed from the cassette. Schlieren can also be caused by inadequate mixing of the gel solution before pouring it into the gel cassette. It is difficult to predict the potency of TEMED unless you know its history of use. TEMED is very hygroscopic and will degrade within six months of purchase if it becomes contaminated with water. Therefore store TEMED in a desiccator at room temperature if you use it frequently, or at 4°C if you use it less than once a week. Cold TEMED must be warmed to room temperature before the bottle is opened to prevent condensation from contaminating the TEMED liquid. 342 Booz
Determine the potency of aps by watching it dissolve, or b listening to it. Weigh out 0.1 g of APS in a small weigh boat, and then place the weigh boat with the aPS onto a dark surface. Add 1 ml of highest purity water directly to the weigh boat, to make a 10% solution If the APS is potent, you will see tiny bubbles fizzing off the surface of the APS crystals. No fizzing is observed with deteriorated APS Or put 0.1 g of APS in a 1.5ml Eppendorf tube and add l ml of water Cap it and listen for the fizzing. If you do not hear little crackling noises, like fizzing, it has lost its potenc and should be replaced Stored solutions of TEMED and aPs may polymerize gels, but if you want to minimize the chance of failure and maximize repro- ducibility, especially with protein gels, prepare APs fresh every day, store TEMED dry at room temperature in a desiccator, and degas your solutions before polymerization. Temperature The temperature of polymerization should be 20 to 22C. If your lab is below 20%C, or if the temperature varies more than five degrees from day to day, reproducibility problems may arise. Note that cold delays polymerization, heat speeds it, and the reaction itself is exothermic What Catalyst Concentration Should You Use? The appropriate catalyst concentration depends on what gel% you are polymerizing. Please refer to Table 12.1 Note that these catalyst concentrations are for protein PAGE gels only Sequencing gels are polymerized differently. The final concentrations of catalysts for a 6 %T sequencing gel, which allow the solution to be introduced into the gel sandwich before polymerization starts, are TEMED, 0.1%(/v), and APS 0.025%(W/v) What Is the Importance of Reagent Purity on Protein Electrophoresis and staining? Reagent purity is extremely important for reproducible results. If the reagents and water you use are very pure, then the polymerization and electrophoresis will be controllable reproducible from day to day. Any problems you have can be ascribed to the sample and its preparation. The following discussion goes into various reagent purity problems and their 343
Determine the potency of APS by watching it dissolve, or by listening to it. Weigh out 0.1 g of APS in a small weigh boat, and then place the weigh boat with the APS onto a dark surface. Add 1 ml of highest purity water directly to the weigh boat, to make a 10% solution. If the APS is potent, you will see tiny bubbles fizzing off the surface of the APS crystals. No fizzing is observed with deteriorated APS. Or put 0.1g of APS in a 1.5 ml Eppendorf tube, and add 1 ml of water. Cap it and listen for the fizzing. If you do not hear little crackling noises, like fizzing, it has lost its potency and should be replaced. Stored solutions of TEMED and APS may polymerize gels, but if you want to minimize the chance of failure and maximize reproducibility, especially with protein gels, prepare APS fresh every day, store TEMED dry at room temperature in a desiccator, and degas your solutions before polymerization. Temperature The temperature of polymerization should be 20 to 22°C. If your lab is below 20°C, or if the temperature varies more than five degrees from day to day, reproducibility problems may arise. Note that cold delays polymerization, heat speeds it, and the reaction itself is exothermic. What Catalyst Concentration Should You Use? The appropriate catalyst concentration depends on what gel % you are polymerizing. Please refer to Table 12.1. Note that these catalyst concentrations are for protein PAGE gels only. Sequencing gels are polymerized differently. The final concentrations of catalysts for a 6 %T sequencing gel, which allow the solution to be introduced into the gel sandwich before polymerization starts, are TEMED, 0.1% (v/v), and APS, 0.025% (w/v). What Is the Importance of Reagent Purity on Protein Electrophoresis and Staining? Reagent purity is extremely important for reproducible results. If the reagents and water you use are very pure, then the polymerization and electrophoresis will be controllable and reproducible from day to day. Any problems you have can be ascribed to the sample and its preparation. The following discussion goes into various reagent purity problems and their resolution. Electrophoresis 343
Table 12. I Gel Percentage vs Catalyst Concentration Gel APS Concentration TEMED Concentration 05% 8-14% 0.05% 0.05% ≥15% 0.05% 0.025% The common contaminants of water are metal ions, especially sodium and calcium, the halide ions, especially chloride, and various organic impurities(Chapter 3 discusses water impurities in greater depth. Each kind of impurity has a different effect; we will not attempt to enumerate all these effects here. Copper ions inhibit acrylamide polymerization, but copper metal and other metals initiate polymerization. Ions can cause ionic interac- tions between the macromolecules in your sample, perhaps causing aggregation of certain proteins, with band smearing the result The organic contaminants can also cause loss of resolution The effects on staining the samples in the gel are also significant, purities in he water can bind the bad background. a detailed discussions about preventing background in a stained gel is provided below. The principle here is that impurities in the water cause problems, and the purest water avail- able should be used for electrophoresis to help prevent these problems. Bacteria in your water purifier can also cause artifacts, such as vertical pinpoint streaks in your gel or on blots stained for total protein Bacteria migrating up the hose from the sink to the filter cartridges is a common cause of contamination note that bacte ria can grow in dishwater left to sit in the sink, so be careful where you place the end of the hose that carries water from the water Another possible source of contamination in your water is the maintenance department in your institution, especially if your water purifier lacks a charcoal filter for removing organic con- taminants. The maintenance department may add organic amine compounds to the distilled water system at your institution to keep scale off the walls of the pipes providing distilled water t your lab. This is commonly done every six months or so. Such contaminants will cause background problems in your stained gels, among other artifacts. The water used to prepare solutions for electrophoresis and staining procedures should be charcoal column-purified and deionized 344 Booz
Water The common contaminants of water are metal ions, especially sodium and calcium, the halide ions, especially chloride, and various organic impurities (Chapter 3 discusses water impurities in greater depth.) Each kind of impurity has a different effect; we will not attempt to enumerate all these effects here. Copper ions inhibit acrylamide polymerization, but copper metal and other metals initiate polymerization. Ions can cause ionic interactions between the macromolecules in your sample, perhaps causing aggregation of certain proteins, with band smearing the result. The organic contaminants can also cause loss of resolution. The effects on staining the samples in the gel are also significant, as impurities in the water can bind the stain, causing bad background. A detailed discussions about preventing background in a stained gel is provided below. The principle here is that impurities in the water cause problems, and the purest water available should be used for electrophoresis to help prevent these problems. Bacteria in your water purifier can also cause artifacts, such as vertical pinpoint streaks in your gel or on blots stained for total protein. Bacteria migrating up the hose from the sink to the filter cartridges is a common cause of contamination. Note that bacteria can grow in dishwater left to sit in the sink, so be careful where you place the end of the hose that carries water from the water purifier. Another possible source of contamination in your water is the maintenance department in your institution, especially if your water purifier lacks a charcoal filter for removing organic contaminants. The maintenance department may add organic amine compounds to the distilled water system at your institution to keep scale off the walls of the pipes providing distilled water to your lab. This is commonly done every six months or so. Such contaminants will cause background problems in your stained gels, among other artifacts. The water used to prepare solutions for electrophoresis and staining procedures should be charcoal column-purified and deionized. 344 Booz Table 12.1 Gel Percentage vs. Catalyst Concentration Gel % APS Concentration TEMED Concentration (w/v) (v/v) 4–7% 0.05% 0.1% 8–14% 0.05% 0.05% 15% 0.05% 0.025%