Transcript SDS-PAGE - Geoscience Research Institute

SDS-PAGE
Sodium Dodecyl Sulfate Polyacrylamid Gel
Electrophoresis
Timothy G. Standish, Ph. D.
©1999 Timothy G. Standish
Sodium 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
SDS
Sodium Dodecyl Sulfate
C12H25NaO4S
H H H H H H H H H H H H O
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 O
Non-polar
Hydrophobic tail
Polar
Hydrophilic head
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
©1999 Timothy G. Standish
SDS and Proteins
SDS
Protein
©1999 Timothy G. Standish
SDS and Proteins
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
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
©1999 Timothy G. Standish
Polyacrylamide Gels
Polyacrilamide is a polymer made of acrylamide
(C3H5NO) and bis-acrilamide (N,N’-methylenebis-acrylamide C7H10N2O2)
CH2 CH
O
C
O
C
NH2
CH2 CH
Acrylamide
Acrylamide
NH2
CH2
O
C
CH2 CH
NH2
Acrylamide
bis-Acrylamide
©1999 Timothy G. Standish
Polyacrylamide Gels
Acrylamide polymerizes in the presence of free
radicals typically supplied by ammonium persulfate
O
C
CH2 CH
O
NH2
C
NH2
CH2 CH
SO4-.
©1999 Timothy G. Standish
Polyacrylamide Gels
l
Acrylamide polymerizes in the presence of free
radicals typically supplied by ammonium persulfate
TMED (N,N,N’,N’-tetramethylethylenediamine)
serves as a catalyst in the reaction
O
C
CH2 CH
O
NH2
C
CH2 CH
O
NH2
C
CH2 CH
O
NH2
C
NH2
CH2 CH
SO4-.
©1999 Timothy G. Standish
Polyacrylamide Gels
bis-Acrylamide polymerizes along with acrylamide
forming cross-links between acrylamide chains
O
C
CH2 CH
O
NH2
C
CH2 CH
O
O
C
C
CH2 CH
CH2 CH
O
NH2
C
NH2
CH2 CH
NH2
CH2
O
NH2
C
CH2 CH
O
NH2
C
CH2 CH
O
NH2
C
NH2
CH2 CH
bis-Acrylamide
©1999 Timothy G. Standish
Polyacrylamide Gels
bis-Acrylamide polymerizes along with acrylamide
forming cross-links between acrylamide chains
©1999 Timothy G. Standish
Polyacrylamide Gels
Pore size in gels can be varied by varying the ratio
of acrylamide to bis-acrylamide
Protein separations typically use a 29:1 or 37.5:1
acrylamide to bis ratio
Lots ofbis-acrylamide
Little
bis-acrylamide
©1999 Timothy G. Standish
SDS-PAGE
1
2
3
3
1
Addition
2 of SDS
Protein becomes rodshaped with uniform
charge distribution
©1999 Timothy G. Standish
©1999 Timothy G. Standish