Polyacrylamide Gel Electrophoresis (PAGE) Gel electrophoresis remains to be a technology in modern-day bioanalytics which cannot be replaced by any other. In the vast majority of cases, Vertical gels are produced from acrylamide which with bis-acrylamide and after the addition of ammonium persulphate (radical donator) and TEMED (catalyst) form a very fine and extremely constant network by means of a radical chain reaction. Application, however, is made more difficult as acrylamide is a very strong nerve poison, the carcinogenic and mutating properties of which have been clearly proven in animal testing. Acrylamide is primarily resorbed through the skin, but above all, it is the absorption of dust in the respiratory tracts or through the facial mucous membrane when weighing out the powder which causes the most serious problems. Rotiphorese® ready-to-use solutions provide the perfect remedy. The solutions, which have been stringently controlled with regard to their acrylamide content and pH, are significantly less dangerous in their application. They are subsequently very easy to use and enable reproducible and high-resolution gel electrophoresis. In our Roth range you will also find all additional reagents needed, such as TEMED, APS, SDS or gel electrophoresis buffers which form a perfect, coordinated team (see below). Rotiphorese® Gel Solutions Ready-to-use acrylamide/bisacrylamide mixtures Rotiphorese® Gel 30 (37.5:1): 30 % acrylamide/bisacrylamide, mixing ratio 37.5:1. Ord.-No. 3029.1 (1 l), 3029.2 (250 ml) Rotiphorese® Gel 40 (19:1): 40 % acrylamide/bisacrylamide, mixing ratio 19:1. Ord.-No. 3030.1 (1 l) Rotiphorese® Gel 40 (29:1): 40 % acrylamide/bisacrylamide, mixing ratio 29:1. Ord.-No. A515.1 (1 l) Rotiphorese® Gel 40 (37.5:1): 40 % acrylamide/bisacrylamide, mixing ratio 37.5:1. Ord.-No. T802.1 (1 l) Acrylamide- and bisacrylamide solution, ready-to-mix Rotiphorese® Gel A: 30 % acrylamide solution. Ord.-No. 3037.1 (1 l) Rotiphorese® Gel B: 2 % bisacrylamide solution. Ord.-No. 3039.1 (1 l), 3039.2 (250 ml) 1 Acrylamide/bisacrylamide mixtures for automated sequencing (fluorescence free) Rotiphorese® NF-acrylamide/bis- solution 40 % (19:1): ready-to-use 40 % acrylamide/bisacrylamide, mixing ratio 19:1. Ord. No. A516.1 (250 ml) Rotiphorese® NF-acrylamide/bis- solution 40 % (29:1): ready-to-use 40 % acrylamide/bisacrylamide, mixing ratio 29:1. Ord. No. A121.1 (250 ml) Rotiphorese® NF-acrylamide/ bis- solution 30 % (29:1): ready-to-use 30 % acrylamide/bisacrylamide, mixing ratio 29:1. Ord. No. A124.1 (250 ml), A124.2 (1 l) Ready-to-use sequencing gel solutions Rotiphorese® sequencing gel concentrate: 25 % acrylamide/bisacrylamide, mixing ratio 19:1 and 50 % urea. Ord. No. 3043.1 (1 l), 3043.2 (100 ml) Rotiphorese® DNA sequencing system (1 l sequencing gel concentrate, 1 l sequencing gel diluent, 250 ml sequencing gel buffer) Ord. No. A431.1 (1 Kit) Recommended Applications: Separation of Nucleic acids Recommended gel solution Gel 40 (3030.1) NF-acrylamide/bis-solution 40 % (A516.1) Sequencing gel concentrate 25 % (3043.1) Gel 40 (A515.1) NF-acrylamide/bis-solution 40 % (A121.1) NF-acrylamide/bis-solution 30 % (A124.1) Gel 30 (3029.1) Gel 40 (T802.1) % C 5 5 5 3.3 3.3 3.3 2.6 2.6 Acrylamide / bisacrylamide 19:1 19:1 19:1 29:1 29:1 29:1 37.5:1 37.5:1 Nucleic acids and proteins Proteins Applications Polyacrylamide gel electrophoresis (PAGE) is used for both high-resolution nucleic acid gels (e.g. sequencing gels) as well as for almost all protein gels. Nucleic acid is, as a rule, separated in a TBE-buffer system, whereas proteins are mixed with SDS for a uniform negative load and separated with Tris/Glycine buffer (SDS-PAGE). A detailed description can be found in e.g. Sambrook and Russel’s Molecular Cloning 3rd Edition, CSHL Press New York, 2004 (Art. No. Y398.1) or in Proteins: Standard Methods of Molecular and Cell Biology, Eckert and Kartenbeck, Springer Verlag Heidelberg, 1997 (Art. No. L937.1). Following tables provide a reference for typical gel mixes in SDS-gel electrophoresis (A) and the separation of nucleic acids (B). A) SDS-PAGE Separation range of SDS-gels Acrylamide concentration (%) Separation range (kD) 6 50-200 8 30-95 10 20-80 12 12-60 15 10-43 2 Resolving gel (data apply to 20 ml gel solution) 30 % acryl- amide mix 40 % acryl- amide mix Gel concentration Aqua dest. (ml) 30 % acrylamide mix (ml) Tris (1.5 M, pH 8.8) (ml) Gel concentration Aqua dest. (ml) 40 % acrylamide mix (ml) Tris (1.5 M, pH 8.8) (ml) 6 % 10.6 4 5 6 % 11.6 3 5 8 % 9.3 5.3 5 8 % 10.6 4 5 10 % 7.9 6.7 5 10 % 9.6 5 5 12 % 6.6 8 5 12 % 8.6 6 5 15 % 4.6 10 5 15 % 7.1 7.5 5 Add in this order: 200 µl 10 % SDS solution (mix carefully, avoid bubbles) 200 µl 10 % ammonia persulphate solution (prepare freshly) 20 µl TEMED (mix carefully, avoid bubbles) Pour gel immediately and overlay with isopropanol Stacking gel (data apply to 5% gels) 30 % acryl- amide mix Gel volume Aqua dest. (ml) 30 % acrylamide mix (ml) Tris (1.0 M, pH 6.8) (ml) SDS (10 % solution) (µl) APS (10 % solution*) (µl) TEMED (µl) Gel volume Aqua dest. (ml) 40 % acrylamide mix (ml) Tris (1.0 M, pH 6.8) (ml) SDS (10 % solution) (µl) APS (10 % solution*) (µl) TEMED (µl) 1 ml 0.68 0.17 0.13 10 10 1 1 ml 0.725 0.125 0.13 10 10 1 3 ml 2.1 0.5 0.38 30 30 3 3 ml 2.185 0.375 0.38 30 30 3 5 ml 3.4 0.83 0.63 50 50 5 5 ml 3.645 0.625 0.63 50 50 5 8 ml 5.5 1.3 1 80 80 8 8 ml 5.84 1 1 80 80 8 10 ml 6.8 1.7 1.25 100 100 10 10 ml 6.3 1.25 1.25 100 100 10 40 % acryl- amide mix * prepare freshly! Be careful to mix the solution thoroughly before and after addition of SDS and TEMED. Avoid bubbles. Pour the stacking gel immediately and insert the comb carefully. 3 B) Separation of nucleic acids Denaturing TBE gels for separation of single stranded nucleic acids (e.g. sequencing gels) (data apply to 100 ml gel solution) 25 % sequencing gel concentrate with urea 30 % acrylamide mix (29:1) Gel concentration Sequencing gel diluent (ml)* 25 % sequencing gel concentrate (ml) Gel concentration Aqua dest. (ad 90 ml) (ml)* 30 % acrylamide mix (ml) Urea (g)** 4 % 74 16 4 % app. 52 13.3 42 4 % app. 55 10 42 6 % 66 24 6 % app. 45 20 42 6 % app. 50 15 42 8 % 58 32 8 % app. 39 26.5 42 8 % app. 45 20 42 40 % Gel concentration acrylamide Aqua dest. (ad 90 ml) (ml)* mix (19:1 or 29:1) 40 % acrylamide mix (ml) Urea (g)** *If required for resolution of secondary structures the gel may be supplemented with formaldehyde by replacing 25 ml aqua dest. with 25 ml formaldehyde. **Results in gels with 42 % urea (7 M) Add in this order: 10 ml 10 x TBE buffer*** (mix carefully, avoid bubbles, degas if required) 400 µl 10 % ammonia persulphate solution (prepare freshly) 50 µl TEMED (mix carefully, avoid bubbles) Pour gel immediately and insert the comb carefully ***Results in 45 % urea if sequencing gel concentrate and sequencing gel diluent are used. If 50% urea are required replace 10 x TBE by the ready-to-use sequencing gel buffer concentrate with 50 % urea (Ord. No. 3050.1). TBE gels for electrophoresis of ds nucleic acid (data apply to 100 ml gel solution) 30 % acrylamide mix (29:1) 40 % acrylamide mix (19:1 or 29:1) Gel concentration Aqua dest. (ml) 30 % acrylamide mix (ml) Gel concentration Aqua dest. (ml) 40 % acrylamide mix (ml) 6 % 69 20 6 % 74 15 10 % 56 33 10 % 64 25 15 % 39 50 15 % 51.5 37.5 Add in this order: 10 ml 10 x TBE buffer (mix carefully, avoid bubbles, degas if required) 1 ml 10 % ammonia persulphate solution (prepare freshly) 60 µl TEMED (mix carefully, avoid bubbles) Pour gel immediately and insert the comb carefully 4 Variable Regulation of Pore Sizes Using Rotiphorese® Gel A and B The pore size of acrylic amide gels can be varied by regulating the total gel concentration (% T) and the percentage of the crosslink (% C). Gels with every desired T/C ratio can be produced with Rotiphorese® Gel A and B: Vt = Total volume of gel casting solution (ml) T = Gel concentration in % = % Acrylamide + % Bisacrylamide C = % Crosslinking = (% Bisacrylamide x 100) / T Va = Volume Gel A in ml Vb = Volume Gel B in ml Applying: Va = (T x (100-C) x Vt) / 3000 Vb = (T x C x Vt) / 200 Example: To prepare 100 ml gel solution with 10 % T and 2.7 % C, calculate as follows: Va = (10 x (100-2.7) x 100) / 3000 = 32.43 ml Gel A Vb = (10 x 2.7 x 100) / 200 = 13.5 ml Gel B Combine 32.43 ml Gel A and 13.5 ml Gel B and fill up the volume to 100 ml with the usually used buffer. Degas and add APS and TEMED, mix thoroughly while avoiding bubbles and pour the gel. Additional Reagents and Solutions: Product TEMED APS Acrylamide p.a., 4 x crist. Bisacrylamide Tris p.a. Glycine p.a. SDS ultra pure Urea Rotiphorese® NF Urea, fluorescence free Rotiphorese® Sequencing Gel Diluent Rotiphorese® Sequencing Gel Buffer Concentrate Rotiphorese® NF 10 x TBE Buffer, fluorescence free Rotiphorese® 10 x TBE Puffer Rotiphorese® 10 x SDS PAGE Buffer Roti®-Stock 20 % SDS Fertiglösung Ord. No. 2367 9592 7906 7867 4855 3908 2326 X999 A120 3047 3050 A118 3061 3060 1057 For further data, safety information, or package sizes please see our catalogue or online at www.carlroth.com Carl Roth GmbH + Co. KG Schoemperlenstraße 3-5 76185 Karlsruhe Postfach 100121 76231 Karlsruhe Telefon: +49 (0) 721/ 5606-0 Telefax: +49 (0) 721/ 5606-149 E-Mail: info@carlroth.com Internet: www.carlroth.com s.s. 07.2012 5
