Protein Structure and Function Lecture 1 Notes: SDS Page and Western Blotting SDS-PAGE Protein Separation There are two commonly used techniques for identifying proteins: (a) SDS-PAGE (b) Western blotting - Gel electrophoresis is used to separate charged molecules via movement through a porous gel. Larger proteins are trapped by the gel matrix, whereas smaller proteins can move easily though the pores. - Polyacrylamide gels are made via polymerisation (catalysed by ammonium persulfate and TEMED) of acrylamide (C3H5NO) and bis- CH NH NH CH acrylamide monomers into a cross-linked HON CH2 CH HON =0 C. ICH network. Acrylamide gels form pores about the -CH CH 0 size of many proteins which enables molecular N,N'-methylenebisacrylamide acrylamide monomer crosslinking monomer ‘sieving' to occur. Polyacrylamide gels are chemically inert and controlling the ammonium persulfate TEMED concentration can easily produce different pore sizes for a variety of proteins (e.g. 10% CH-CH- CH-CH2 CH-CH2 CH-CH acrylamide is useful for a molecular weight range ¢=0 C=O 1 C=O C=0 NH2 NH NH2 NH2 of 20-300kDa). CH2 .- crosslink NH2 ?=0 NH NH2 ?=0 NH2 ¢=0 CH-CH - Native gels (aka non-denaturing gels) separate folded proteins via a matrix of polyacrylamide polymer fibres. Protein separation depends on a combination of size and charge. C=0 CH-CH2 CH-CH2 CH-CH, polyacrylamide - However, the net charge of natively folded proteins is determined by amino acid composition therefore different proteins with the same molecular weight migrate at different speeds in an electrical field depending on their charge and 3D shape. Thus, SDS is used to separate proteins based on molecular weight only (i.e. by denaturing the protein and masking the intrinsic net charge of the protein). SDS-PAGE gels are more commonly used than native gels. - SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) separates proteins based on molecular weight via differential rates of migration (using an electrical field) through a sieving matrix (gel)
- Proteins are coated with SDS (sodium dodecyl sulfate, NaC12H25SO4), a negatively charged detergent present in the sample buffer. The sample is boiled and a reducing agent (usually DTT or B-ME/B- mercaptoethanol) breaks down disulphide bonds between proteins and disrupts the tertiary structure (makes the proteins linear molecules). SDS coats the proteins with a uniform negative charge, masking the intrinsic charges of the R-groups as it binds (stoichiometrically) fairly uniformly to linear proteins (around 1.4g SDS per 1g of protein) meaning the charge of the protein is approximately proportional to its molecular weight. SDS-coated proteins Place mixture of proteins on gel, apply electric field Cross-linked polyacrylamide Partially separated proteins gel Direction of migration + Stain to visualize separated bands à - SDS-coated proteins are linear molecules 18Å wide and have a length proportional to their molecular weight, so the molecular radius and thus mobility in the gel is determined by the protein's molecular weight. There will be no differential migration based on charge because the SDS-coated proteins have an (almost) equal charge to mass ratio. - Laemmli electrophoresis is a discontinuous (i.e. the buffer and gel tank are different) buffer