蛋白质电泳技术

. Original citation: Schagger, H. and G. von Jagow. 1987. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166: 368-379. Reagents: Anode Buffer (+): 200 mM Tris pH 8.9 (can dilute from 10X stock) Cathode Buffer (-): 100 mM Tris/100 mM Tricine/0.1% SDS (no need to pH, but will be ~8.25) these can be made as 10X stocks and diluted before use Gel Buffer: 3.0 M Tris, pH 8.45/0.3% SDS Note: the pHs given for the anode and gel buffers are essential Stacking acrylamide: 48% acrylamide/1.5% bis-acrylamide Separating acrylamide: 46.5% acrylamide/1.5% bis-acrylamide Sample buffer: (add equal volume to sample), for 20 ml:5 ml 0.5 M Tris, pH 6.8 4 ml 20% SDS 1 ml 2-mercaptoethanol 4 ml 50% glycerol 0.004 g bromophenol blue 6 ml waterPouring Gels For each minigel (Hoeffer Mighty Small or BioRad Mini Protean-II--scale up as required): 1. Separating gel: 15% gel 2 ml separating acrylamide 2 ml gel buffer 2 ml 50% glycerol 10% gel 1.22 ml separating acrylamide 2 ml gel buffer 2 ml 50% glycerol 0.78 ml waterpolymerize with 75 ul 10% APS and 7.5 ul TEMED 2. Stacking gel:0.25 ml stacking acrylamide 0.75 ml gel buffer 2.0 ml water 20 ul 10% APS 2 ul TEMED Polymerize both the stacking and separating gels at the same time (I used small disposable tubes), and pour stacking gel directly onto the separating gel (pour carefully but quickly--they wont mix but the separating gel polymerizes within 2-3 minutes). Make each separating gel mixture separately and add TEMED and APS right before pouring. Multiple stacking gel mixtures can be made in the same tube, but you have around 10 minutes before these start to polymerize too. Sample loading and electrophoresis: For the minigels, 5-10 ul per well gives best results. Layer the samples in the well very carefully, and be sure to flush out any unpolymerized acrylamide before loading. Electrophorese at 25-35 mA (constant current) per gel in minigel setup. Foam will appear on the top (cathode), and additional cathode buffer may have to be added during the run. 15% tricine-PAGESeparation of prepilin and processed pilin from P. aeruginosa on a Coomassie-blue stained tricine-15% PAGE gel. The two forms differ by 6 amino acids after cleavage by[1] [2] [3] [4] 下一页
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