Sodium Dodecylsulfate
Polyacrylamide Gel
Electrophoresis(SDS-PAGE)
BY: JANE LOH
JENNY ONG
Overview
a. Introduction
b. Principle of Sds-page
c. Gel preparation
d. Process of Sds-page
e. Visualization of protein
f. Applications
g. Advantages and disadvantages
h. Conclusion
i. References
Introduction
Standard test that used to determine
the charged molecules, mainly proteins
and nucleic acids.
Widely used in
biochemistry, forensics, genetics and
molecular biology.
Laemmli system of SDS-PAGE was
first introduced in 1970s.
Principle
Separates protein in an electric field
Migrate through a liquid or semisolid
medium when subjected to an electric
field from anode to cathode terminal
Molecules flow at different rates
depend on the molecular size of
proteins
Sds-coated large proteins migrate slowly through the gel
matrix and small proteins migrate quickly through the matrix
The nearer the band to the well, the larger the molecular
size of protein
What is SDS?
negatively charged detergent sodium dodecylsulfate
(SDS)
used to denature and linearize the proteins
coated the proteins with negatively charged
What is PAGE?
SDS-PAGE is differentiated into two
systems
Continuous sds-page
Discontinuous sds-page
Polyacrylamide is used to form a gel,
a matrix of a pores which allow the
molecules migrate at different rates.
Polyacrylamide gel
The size of pores is determined by the
concentration of acrylamide.
The higher the concentration, the
smaller the size of pores.
Discontinuos Sds-Page consists of
two different gels.
Stacking gel (top gel)-4% of acrylamide
Separating gel (bottom gel)- range from
5 to 15%of acrylamide
Why polyacrylamide used for a
gel?
Chemically inert
Electrically neutral
Hydrophillic
Transparent for optical detection
Preparation of GEL
1. Clean the plates and combs.
2. Set-up the plates on the rack.
3. Pour the separating gel.
4. Pour the stacking gel.
5. Gel storage.
Process of SDS-PAGE
1. Boil the samples for 10 minutes to
completely denatures the proteins.
2. Assemble the gel into the apparatus.
3. Pour the buffer solution into the
chamber.
4. Load 20uL of samples into the well.
5. After that, run electrophoresis by
connecting the current supplies.
Visualization of protein bands
Visualizes the band under UV light
Types of Stains:
1. Coomassie Blue
Traditional method requires staining followed by
destaining to remove background gel staining.
Most common and least sensitive
Limited to ~100ng of protein
2. Silver Stain
o Most sensitive test
o Detection limit: 0.1-1.0ng of protein
A. Staining band with Western blot; B. Coomassie blue stain; C.
Silver stain
Applications
Determine purity of protein samples
Determine molecular weight of protein
Identifying disulfide bonds between
protein
Quantifying proteins
Blotting applications
Advantages & disadvantages
Advantages Disadvantages
Migration is proportional to Poor band resolution due to
the molecular weight high alkaline operating pH
Highly sensitive test, Acrylamide gel is potent
separates 2% difference in neurotoxin chemical
mass
Require small amount of Gel preparation is difficult and
samples require longer time
Stable chemically cross- Highly cost
linked gel
Video of SDS-PAGE
Procedure
Source origin: Labtricks.com
Conclusion
Sds-page is a technique that used to
separate proteins according to their
molecular size through the gel.
Proteins are unfolded and migrate
from cathode to anode terminal at
different rates.
Molecular weight is determined by
compare the result with a standard
curve of relative motility of standard
proteins
References
Introduction to Agarose and
Polyacrylamide Gel Electrophoresis
Matrices, written by Patricia Barril and
Silvia Nates
Discontinuous polyacrylamide gel
eletrophoresis of DNA fragments.
Methods in molecular biology. Written
by Budowle, B. Allen, 1991.
Questions
1) SDS-PAGE has been used
(a)To analyze different types of proteins in a biological sample
(b)To characterize membrane proteins in their native conformation
(c)To characterize the enzyme activities in the lysosome
(d)To isolate nuclear DNA
2) In an SDS-PAGE experiment, proteins are separated on
the basis of their
3) In an SDS-PAGE gel
(a) Proteins are denatured by SDS
(b)Proteins have the same charge to mass ratio
(c)Smaller proteins migrate more rapidly through the gel
(d)All the above
