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SDS-Page Technique Sodium Dodecyl Sulfate Polyacrylamide Laboratory of Molecular Biology and Endemic Diseases Created by: Lara Riguetti Introduction SDS-PAGE separates molecules according to your molecular weight, based on their differential rates of migration through a sieving matrix (a gel) under the influence of an electric field applied. 1 2 3 4 Estudar para explicar sobre a imagem. Principle SDS coats the protein with a uniform negative charge, which masks the intrinsic charges on the R-groups. Role of SDS Denaturant Gel X Native Gel Adicionar explicação sobre o mercaptoetanol. Polyacrylamide Gel A mixture of two polymers: acrylamide and bis-acrylamide. The acrylamide is a linear molecule, while the BIS-acrylamide is present in the form of "T". Destacar a acrilamida e a bis dentro da imagem. TEMED and APS Ammonium Persulfate and TEMED are the initiators of the polymerization. APS (Ammonium Persulfate) TEMED (N,N,N’,N’ - tetramethylethane - 1,2 - diamine) Falar o nome Amônio de Persulfato. The gel used is divided in Buffer system and gel steps Tris-Gly ph 8.3 Tris-HCL ph 6.8 Tris-HCL ph 8.8 What happens in the stacking and running gels? Stacking Gel Running Gel Migration The molecules migrates differently along the gel. Tomar cuidado com a expressão ”correm mais rápido” After the submission of electrophoresis, the proteins "laid down" to gel with acetic acid. The most commonly used dyes having different protein detection limits are: Silver Stain Kit Coomassie Brilliant Blue R-250 G-250 Silver Stain Coomassie Blue
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