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
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