I am HAFIZ M WASEEM FROM mailsi vehari BSc in science college Multan Pakistan MSC university of education Lahore Pakistan i love Pakistan and my teachers

1. HAFIZ
MUHAM
MAD
WASEEM
BA BA G
LAHORE

2. Advanced Analytical Techniques
UNIVESITY OF EDUCATION
LAHORE PAKISTAN

3. Introduction
What is SDS-PAGE?
 Separation of macromolecules
on the basis of their electro-
phoretic mobility is
called electrophoresis
 The techniques cannot be used
to determine molecular weight
of biological molecules because
the mobility of a substance in
the gel depends on both charge
and size
 So, there is a need to denature
the proteins with some
detergent like SDS so that they
lose their secondary, tertiary or
quaternary structures and
become linear

4. Introduction
 This method is called sodium
dodecyl sulfate
polyacrylamide gel
electrophoresis (SDS-PAGE)
 The method is also called
Laemmli method after
Laemmli who was the first to
publish a paper employing
SDS-PAGE
 A polypeptide chain binds
with SDS in proportion to its
relative molecular mass
 This results in fractionation
by approximate size during
electrophoresis

5. Introduction
 Negative charges on SDS
destroy most of the complex
structure of proteins
 These molecules are strongly
attracted toward an anode in
an electric field
 Polyacrylamide gels restrain
larger molecules from
migrating as fast as the
smaller molecules
 SDS-PAGE is the most widely
used analytical method in
biochemistry and molecular
biology
 Characterization of proteins is
also possible

6. Advanced Analytical Techniques
SDS-Polyacrylamide
Gel Electrophoresis-
Principle

7. Principle of SDS-PAGE
 SDS denatures the proteins by
binding to hydrophobic
regions
 Non-covalent bonds are
disrupted and the proteins
acquire a net negative charge
 A Concurrent treatment with
a disulfide reducing agent
such as β-mercaptoethanol or
DTT (dithiothreitol) also
denature the proteins by
reducing disulfide linkages
 Proteins samples get uniform
structure and charge
 A suitabe dye is used to
monitor electrophoresis

8.  Separation depends on their
molecular weight only
 Small proteins migrate faster
than the larger ones through
the gel matrix under the
influence of the applied
electric field
 Number of SDS molecules that
bind is proportional to the
size of the protein
 So, the SDS-treated proteins
get similar charge-to-mass
ratios and similar shapes
move towards anode and
separate only according to
their molecular weight
Principle of SDS-PAGE

9. Advanced Analytical Techniques

10.  Sample containing proteins or
nucleic acids is prepared by
using
 Homogenizer
 Sonicator
 Filtration & centrifugation
 It is mixed with some suitable
denaturant like SDS for
proteins
 Proteins are also heated with a
reducing agent like Beta-
mercaptoethanol which
denatures the proteins by
reducing disulfide linkages
thus breaking the complex
structures
Procedure of SDS-PAGE
Sample Preparation

11.  SDS-PAGE gels consist of
separating and stacking gels
 For separating gel prepare gel
solution (desired %age) and
pour it into the gap between
the glass plates
 It is degassed under a vacuum
or butanol is added to prevent
the formation of air bubbles
during polymerization
 APS and TEMED are added to
initiate polymerization
 Wash top of the gel for several
times to remove acrylamide
that is unpolymerized
Procedure of SDS-PAGE
Preparing the gel

12.  Stacking gel (5%) is poured on
top of the separating gel and a
comb is inserted to make wells
 After polymerization the comb
is removed which is ready for
electrophoresis
 Acrylamide concentration may
range from 5% to 25%
 Lower percentage gels are
better for resolving very high
molecular weight molecules
 While higher percentages are
needed to resolve smaller
proteins
 Finally the gel is fixed in the
electrophoresis chamber
Procedure of SDS-PAGE
Preparing the gel

13.  Samples are mixed with
loading buffer and are loaded
in the wells
 The ladder is loaded in the
first well
 Various buffer systems are
used in PAGE depending on
the nature of the samples
 Generally the gel is run for 1
hour at 120V voltage for 12%
separating gel
 Negatively charged proteins
or nucleic acids migrate
towards the anode
 Smaller molecules move
faster than the larger ones
Procedure of SDS-PAGE
Electrophoresis

14.  After electrophoresis, the
gel is stained with dyes like
Coomassie Blue
 Different Proteins are
stained as distinct bands in
the gel
 Different proteins are
stained differently with the
stains
 Detergents like SDS are used
to separate the folded
proteins
 A suitable marker is used to
estimate molecular mass of
the unknown proteins
Procedure of SDS-PAGE
Staining & Visualization

15. Advanced Analytical Techniques

16.  A number of factors affect
the rate of electrophoresis
like
 Concentration of gels
 Size of molecules being
electrophoresed
 Voltage used
 Time
 Ionic strength of
buffers
 Dyes such as ethidium
bromide used during
electrophoresis
 Characterization of proteins
by Western blotting by
transferring to a membrane
Factors affecting SDS-P AGE

17. Advanced Analytical Techniques

18.  SDS-PAGE has a number of
applications which include
• Measuring molecular
weights of different bio-
molecules
• Estimation of purity of the
proteins
• Quantification of proteins
• Analysis of number and
size of proteins subunits
• Characterization of
proteins by Western
blotting
• Staining of proteins
• Labeling of different
proteins
• Protein mapping
Applications of SDS-PAGE