Biotechnology. Lysozyme An enzyme that cleave the bonds in the cell membrane Lyses the cell membrane: releases proteins - mercaptoethanol

Biotechnology

LysozymeAn enzyme that cleave the bonds in the cell membraneLyses the cell membrane: releases proteins

- mercaptoethanolReduces di-sulfide bridges in proteins

SDSDiscussed later!!!

Resuspend Cells in TE Buffer

Add 2x sample buffer

Add lysozyme (5mg/ml) into select vials

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

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

O

CHCH2

NH2C

O

CHCH2

NH2C

SO4-.

Acrylamide polymerizes in the presence of free radicals typically supplied by ammonium persulfate

Acrylamide Acrylamide

Free radicals

SO4-.

O

CHCH2

NH2C

O

CHCH2

NH2CNH2

O

CHCH2

C

O

CHCH2

NH2C

In the meantime TEMED serves as a catalyst

bis-Acrylamide polymerizes along with acrylamide forming cross-links between acrylamide chains

O

CHCH2

NH2C

O

CHCH2

NH2C

O

CHCH2

NH2CNH2

O

CHCH2

C

O

CHCH2

NH2C

O

CHCH2

NH2C

bis-Acrylamide

O

CH

CH2

NH2C

O

CHCH2

NH2C

CH2

Stacking gelLarge pore sizeStacks proteins

Separating gelSmall pore sizeSeparates proteins base on molecular weight

spacers

comb

interface

Stacking gel

Separating gel

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 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.

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

Sample buffer waste- contains a hazardous reducing agent, Beta mercaptoethanol -stinks like rotten eggsEppendorf tubes should be thrown into the

sample buffer waste-container