biotechnology. lysozyme an enzyme that cleave the bonds in the cell membrane lyses the cell...
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![Page 1: Biotechnology. Lysozyme An enzyme that cleave the bonds in the cell membrane Lyses the cell membrane: releases proteins - mercaptoethanol](https://reader036.vdocuments.us/reader036/viewer/2022062407/56649e0d5503460f94af6c1a/html5/thumbnails/1.jpg)
Biotechnology
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LysozymeAn enzyme that cleave the bonds in the cell membraneLyses the cell membrane: releases proteins
- mercaptoethanolReduces di-sulfide bridges in proteins
SDSDiscussed later!!!
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Resuspend Cells in TE Buffer
Add 2x sample buffer
Add lysozyme (5mg/ml) into select vials
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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
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Polymerization of acrylamide and bisacrylamide monomers is Induced by ammonium persulfate (APS) Catalyzed by TEMED.
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O
CHCH2
NH2C
O
CHCH2
NH2C
SO4-.
Acrylamide polymerizes in the presence of free radicals typically supplied by ammonium persulfate
Acrylamide Acrylamide
Free radicals
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SO4-.
O
CHCH2
NH2C
O
CHCH2
NH2CNH2
O
CHCH2
C
O
CHCH2
NH2C
In the meantime TEMED serves as a catalyst
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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
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Stacking gelLarge pore sizeStacks proteins
Separating gelSmall pore sizeSeparates proteins base on molecular weight
spacers
comb
interface
Stacking gel
Separating gel
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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
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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.
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In aqueous solutions, SDS polarizes releasing Na+ and retaining a negative charge on the sulfate head
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Sample buffer waste- contains a hazardous reducing agent, Beta mercaptoethanol -stinks like rotten eggsEppendorf tubes should be thrown into the
sample buffer waste-container