Course: SAT 170 Introductory Biotechnology

Unit 5 Reading and Multimedia Assignment

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“Proteomics/Protein Separations- Electrophoresis/Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)” by Wikibooks, CC BY SA 3.0

http://en.wikibooks.org/wiki/Proteomics/Protein_Separations-_Electrophoresis/Sodium_dodecyl_sulphate_polyacrylamide_gel_electrophoresis_(SDS-PAGE)

SDS Page

Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)is a method of gel elctrophoresis to separate proteins based on the their mass.The proteins are dissolved in sodium dodecyl sulfate (SDS),a detergent that breaks up the interactions between proteins,and then electrophorised. The smallest molecules move through the gel faster, while larger molecules take longer and result in bands closer to the top of the gel.

Gel

The gel used for SDS-PAGE is made out of acrylamide which form crooss-linked polymers of polyacrylamide. Standard gels are typically composed of two layers, one top-most layer called the stacking gel and a lower layer called separating or resolving gel.The stacking layer contains a low percentage of acylamide and has low pH, while the acrylamide concentration of the separating gel varies according to the samples to be run and has higher pH.The difference in pH and acrylamide concentration at the stacking and separating gel provides better resolution and sharper bands in the separating gel.

Procedure

The samples are treated with SDS (sodium dodecyl sulfate),an anionic detergent which denatures the protein by breaking the disulfide bonds and gives negative charge to each protein in proportion to its mass. Without SDS,different proteins with similar molecular weights would migrate differently due to differences in folding, as differences in folding patterns would cause some proteins to better fit through the gel matrix than others.SDS linearizes the proteins so that they may be separated strictly by molecular weight. The SDS binds to the protein in a ratio of approximately 1.4 g SDS per 1.0 g protein, giving an approximately uniform mass:charge ratio for most proteins, so that the distance of migration through the gel can be assumed to be directly related to only the size of the protein. Proteins may be further treated with reducing agent, such as dithiothreitol(DTT) or TRP(Tributyl phosphine)to break any reformed disulfide bonds and then alkalated with iodoacetamide to prevent reformation of disulfide bonds. A tracking dye like bromophenol blue may be added to the protein solution to track the progress of the protein solution through the gel during the electrophoretic run.

Two SDS-PAGE-gels after a completed run

Electrophoresis

The proteins denatured by SDS are applied to one end of a layer of polyacrylamide gel submerged in a buffer. Buffers provide uniform pH and ions for conducting electric potential. When an electric current is applied across the gel, the negatively-charged proteins migrate across the gel to the positive pole. Short proteins will more easily fit through the pores in the gel and move fast, while larger ones will have more difficulty. Due to differential migration based on their size, smaller proteins move farther down the gel, while larger ones stay closer to the point of origin. After a given period of time, proteins might have separated roughly according to their sizes. Proteins of known molecular weight (marker proteins) can be run in a separate lane in the gel to calibrate the gel.

Staining and analysis

Following electrophoresis, the gel may be stained with Coomassie Brilliant Blue or silver stain to visualize the separated proteins. After staining, different proteins will appear as distinct bands within the gel according to their sizes and molecular weights. The molecular weight of a protein in the band can be estimated by comparing it with the marker proteins of known molecular weights. The separated proteins can be cut from the gel and further analyzed by other proteomics techniques.