SDS-PAGE

Sodium Dodecyl Sulfate -

Polyacrylamid Gel
Electrophoresis

©1999 Timothy G. Standish

 Sodium Dodecyl Sulfate
SDS is a common ingredient in detergents
Other names for SDS include laurel sulfate and
sodium laurel sulfate
As a detergent SDS destroys protein secondary,
tertiary and quaternary structure
This makes proteins rod shaped

©1999 Timothy G. Standish

 Sodium Dodecyl Sulfate
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 Polar
Hydrophobic tail 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 quaternary structure
Thus shape is no longer an issue as the protein SDS
complex becomes rod shaped

©1999 Timothy G. Standish

 SDS 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

©1999 Timothy G. Standish

 Polyacrylamide Gels
Polyacrylamide is a polymer made of acrylamide
(C3H5NO) and bis-acrylamide (N,N’-methylene-
bis-acrylamide C7H10N2O2)

CH2 CH Acrylamide
O
O C NH2
C NH2
CH2
CH2 CH
O C NH2
Acrylamide
Acrylamide CH2 CH

bis-Acrylamide
©1999 Timothy G. Standish
 Polyacrylamide Gels
Acrylamide polymerizes in the presence of free
radicals typically supplied by ammonium persulfate

O O

C NH2 C NH2
CH2 CH CH2 CH

SO4-2

©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 O O O

C NH2 C NH2 C NH2 C NH2

CH2 CH CH2 CH CH2 CH CH2 CH

SO4-2
©1999 Timothy G. Standish
 Polyacrylamide Gels
bis-Acrylamide polymerizes along with acrylamide
forming cross-links between acrylamide chains
O O O

C NH2 C NH2 C NH2

CH2 CH CH2 CH CH2 CH CH2 CH

O C NH2

CH2 O O O

O C NH2 C NH2 C NH2 C NH2

CH2 CH CH2 CH CH2 CH 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
2
1
Addition of SDS:
Protein becomes rod-shaped
with uniform charge
distribution ©1999 Timothy G. Standish
©1999 Timothy G. Standish