1. Protein Electrophoresis I
Biotechnology

2. 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!!!

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

4. 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.

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

6. 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

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

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

9. 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

10. 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

11. 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.

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

13. 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