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©1999 Timothy G. Standish SDS-PAGE SDS-PAGE Sodium Dodecyl Sulfate Sodium Dodecyl Sulfate - Polyacrylamid Gel - Polyacrylamid Gel Electrophoresis Electrophoresis Timothy G. Standish, Ph. D.
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©1999 Timothy G. Standish
SDS-PAGESDS-PAGESodium Dodecyl Sulfate - Sodium Dodecyl Sulfate -
Polyacrylamid Gel Polyacrylamid Gel ElectrophoresisElectrophoresis
Timothy G. Standish, Ph. D.
©1999 Timothy G. Standish
Sodium Dodecyl SulfateSodium Dodecyl Sulfate SDS is a common ingredient in detergents Other names for SDS include lauryl sulfate and sodium lauryl
sulfate As a detergent SDS destroys protein secondary, tertiary and
quaternary structure This makes proteins rod shaped SDS also sticks to proteins in a ratio of approximately 1.4 g of
SDS for each gram of protein Negative charge on the sulfate groups of SDS mask any charge
on the protein
©1999 Timothy G. Standish
PolarHydrophilic head
Non-polarHydrophobic tail
SDSSDSSodium Dodecyl SulfateSodium Dodecyl Sulfate
Because it is amphipathic, SDS is a potent detergent SDS may also be called sodium lauryl sulfate and is a common
ingredient of shampoos, laundry detergent and other cleaning products
H-C-C-C-C-C-C-C-C-C-C-C-C-O-S-O-Na+
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
O
O
C12H25NaO4S
©1999 Timothy G. Standish
SDS and ProteinsSDS and Proteins
SDS
Protein
©1999 Timothy G. Standish
SDS and ProteinsSDS and Proteins
In aqueous solutions, SDS polarizes releasing Na+ and retaining a negative charge on the sulfate head
So much SDS binds to proteins that the negative charge on the SDS drowns out any net charge on protein side chains
In the presence of SDS all proteins have uniform shape and charge per unit length
SDS nonpolar chains arrange themselves on proteins and destroy secondary tertiary and quarternary structrure
Thus shape is no longer an issue as the protein SDS complex becomes rod shaped
©1999 Timothy G. Standish
Acrylamide
Acrylamide
Polyacrylamide GelsPolyacrylamide Gels Polyacrilamide is a polymer made of acrylamide
(C3H5NO) and bis-acrilamide (N,N’-methylene-bis-acrylamide C7H10N2O2)
O
CH
CH2
NH2C
O
CHCH2
NH2C
CH2
bis-Acrylamide
O
CHCH2
NH2C
Acrylamide
©1999 Timothy G. Standish
Polyacrylamide GelsPolyacrylamide Gels
O
CHCH2
NH2C
O
CHCH2
NH2C
SO4-.
Acrylamide polymerizes in the presence of free radicals typically supplied by ammonium persulfate
©1999 Timothy G. Standish
Polyacrylamide GelsPolyacrylamide Gels Acrylamide polymerizes in the presence of free
radicals typically supplied by ammonium persulfate
SO4-.
O
CHCH2
NH2C
O
CHCH2
NH2CNH2
O
CHCH2
C
O
CHCH2
NH2C
TMED (N,N,N’,N’-tetramethylethylenediamine) serves as a catalyst in the reaction
©1999 Timothy G. Standish
Polyacrylamide GelsPolyacrylamide Gels bis-Acrylamide polymerizes along with acrylamide
forming cross-links between acrylamide chainsO
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
©1999 Timothy G. Standish
Polyacrylamide GelsPolyacrylamide Gels bis-Acrylamide polymerizes along with acrylamide
forming cross-links between acrylamide chains
©1999 Timothy G. Standish
Polyacrylamide GelsPolyacrylamide Gels Pore size in gels can be varied by varying the ratio
of acrylamide to bis-acrylamide
Lots of bis-acrylamideLittle bis-acrylamide
Protein separations typically use a 29:1 or 37.5:1 acrylamide to bis ratio
©1999 Timothy G. Standish
1 2 3
SDS-PAGESDS-PAGE
Addition of SDS23
1 Protein becomes rod-shaped with uniform charge distribution
©1999 Timothy G. Standish
