Protein Electrophoresis I Biotechnology

Key Reagents Lysozyme An enzyme that cleave the bonds in the cell membrane Lyses the cell membrane: releases proteins b- mercaptoethanol Reduces di-sulfide bridges in proteins SDS Discussed later!!!

1) Lysis of Cells Resuspend Cells in TE Buffer Add 2x sample buffer Add lysozyme (5mg/ml) into select vials

2) SDS-Page Polyacrylamide gel electrophoresis (PAGE) is the most common analytical technique used to separate and characterize proteins. Matrix for separation is polyacrylamide: a polymer of acrylamide crosslinked with bis- acrylamide PAGE Polyacrylamide gel electrophoresis (PAGE) is probably the most common analytical technique used to separate and characterize proteins. A solution of acrylamide and bisacrylamide is polymerized. Acrylamide alone forms linear polymers. The bisacrylamide introduces crosslinks between polyacrylamide chains. The 'pore size' is determined by the ratio of acrylamide to bisacrylamide, and by the concentration of acrylamide. A high ratio of bisacrylamide to acrylamide and a high acrylamide concentration cause low electrophoretic mobility. Polymerization of acrylamide and bisacrylamide monomers is induced by ammonium persulfate (APS), which spontaneously decomposes to form free radicals. TEMED, a free radical stabilizer, is generally included to promote polymerization. SDS PAGE Sodium dodecyl sulfate (SDS) is an amphipathic detergent. It has an anionic headgroup and a lipophilic tail. It binds non-covalently to proteins, with a stoichiometry of around one SDS molecule per two amino acids. SDS causes proteins to denature and dissassociate from each other (excluding covalent cross-linking). It also confers negative charge. In the presence of SDS, the intrinsic charge of a protein is masked. During SDS PAGE, all proteins migrate toward the anode (the positively charged electrode). SDS-treated proteins have very similar charge-to-mass ratios, and similar shapes. During PAGE, the rate of migration of SDS-treated proteins is effectively determined by molecular weight.

Key Reagents Polymerization of acrylamide and bisacrylamide monomers is Induced by ammonium persulfate (APS) Catalyzed by TEMED.

Polymerization Acrylamide polymerizes in the presence of free radicals typically supplied by ammonium persulfate Acrylamide Acrylamide O CH CH2 NH2 C O CH CH2 NH2 C SO4-. Free radicals

Polymerization In the meantime TEMED serves as a catalyst NH2 O CH CH2 SO4-.

Polymerization bis-Acrylamide polymerizes along with acrylamide forming cross-links between acrylamide chains O CH CH2 NH2 C bis-Acrylamide

Polyacrylamide Gel Stacking gel Large pore size Stacks proteins Separating gel Small pore size Separates proteins base on molecular weight spacers comb interface Stacking gel Separating gel

Key Reagent Sodium dodecyl sulfate (SDS) is an amphipathic detergent. SDS may also be called sodium lauryl sulfate and is a common ingredient of shampoos laundry detergent and other cleaning products SDS is highly charged and binds to protein in proportion to their molecular weight Exert strong internal replusive forces that tend to stretch out the random coil protein into a rod-like shape

Key Reagent: SDS SDS is a negatively charged detergent. ‘Masks’ charge on protein so that all proteins act the same regardless of charge. confers negative charge. the intrinsic charge of a protein is masked. Due to the stoichiometric binding of SDS to proteins and the overall negative charge all protein will migrate toward the anode (the positively charged electrode) in a size dependent manner Prevents protein shape from influencing gel run. Disrupts secondary and tertiary protein structures by breaking hydrogen bonds and unfolding protein.

Key Reagent: SDS In aqueous solutions, SDS polarizes releasing Na+ and retaining a negative charge on the sulfate head

Caution Sample buffer waste- contains a hazardous reducing agent, Beta mercaptoethanol - stinks like rotten eggs Eppendorf tubes should be thrown into the sample buffer waste-container