Sodium Dodecyl Sulfate

Polyacrylamide gel
electrophoresis
(SDS-PAGE)
SDS-PAGE
 A technique widely used to separate
proteins according to their electrophoretic
mobility


 SDS gel electrophoresis of samples having

identical charge per unit mass due to
binding of SDS results in fractionation by
size and is probably the world's most
widely used biochemical method
SODIUM DODECYL SULFATE
 C12 H25 NaO4S
 MW: 288.38

 most common dissociating agent used to
denature native proteins to
individual polypeptides


 When a protein mixture is heated to 100 °C

in presence of SDS, the detergent wraps
around the polypeptide backbone. It binds
to polypeptides in a constant weight ratio
of 1.4 g/g of polypeptide
TISSUE PREPARATION
TISSUE PREPARATION
 Samples may be taken from whole tissue or
cell culture
 Combination of biochemical and mechanical
techniques can be used to separate
different cell compartments and organelles
 The solution of proteins to be analyzed is
mixed with SDS, an
anionic detergent which denatures
secondary and non–disulfide–linked tertiary
structures, and applies a negative charge
to each protein in proportion to its mass

TISSUE PREPARATION
 A tracking dye may be added to the protein
solution (of a size smaller than protein) to
allow the experimenter to track the
progress of the protein solution through
the gel during the electrophoretic run
PREPARING ACRYLAMIDE
GELS
ELECTROPHORESIS
STAINING
 the gel may be stained allowing
visualization of the separated proteins, or
processed further
 different proteins will appear as distinct
bands within the gel. It is common to
run molecular markersof known molecular
weight in a separate lane in the gel, in
order to calibrate the gel and determine
the weight of unknown proteins by
comparing the distance traveled relative to
the marker
STAINING
 The gel is actually formed because the
acrylamide solution contains a small
amount.
 Gel electrophoresis is usually the first choice
as an assay of protein purity due to its
reliability and ease. The presence of SDS
and the denaturing step causes proteins to
be separated solely based on size. False
negatives and positives are possible. A
comigrating contaminant can appear as
the same band as the desired protein.