Protein Electrophoresis

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Experiment

Separation of main plasma protein


by using SDS-PAGE

Prepared by
Eman Alshehri
Objectives
 Separation of plasma protein and determine its
MWt.

 To learning some valuable skills in the field of


separation of protein by SDS-PAGE.

 Identify the Varied uses of this technique.


Introduction

SDS-PAGE (Sodium dodecyle sulfate polyacrylamid


gel electrophoresis) is method separates proteins
based primarily on their molecular weight in electric
field by uses a discontinuous polyacrylamide gel as
a support medium and SDS to denature the proteins
Principle
• Proteins are globular in secondary and tertiary structure
due to disulfide bonds, hydrophobic interactions and
hydrophilic interactions with their aqueous environment.
Therefore, something must be done to break the
secondary and tertiary structure of the proteins in the
sample for accurate analysis of peptide size to occur.
• Sodium dodecyl sulfate (SDS) is a detergent possessing
both a hydrophobic end (the dodecyl group) and a
hydrophilic end (the sulfate group).
• The tertiary structure of most proteins
often relies upon hydrophobic interactions
at the core of the protein. The hydrophobic
end of SDS breaks these interactions
through interactions with the hydrophobic
side chains of the amino acids. Similarly, a
sulfate group can disrupt hydrogen
bonding in secondary protein structure .
• Disulfide bonds holding tertiary or quaternary structure together can
be broken by using a reducing agent, such as beta-mercaptoethanol
(BME).
• Finally, heating the protein sample also aids the denaturation and
unfolding process allowing chemicals like SDS and BME to interact
with the protein.
• In addition to denaturing the protein, SDS also serves an additional
purpose. Because each protein is coated with SDS molecules and
the charge. This means that when an electrical field is applied to the
gel in buffer, each protein molecule will move toward the positive
electrode. This allows the acrylamide to separate the proteins based
on size
• SDS disrupts the secondary, tertiary and
quaternary structure of the protein to
produce a linear polypeptide
• chain coated with negatively charged SDS
molecules. 1.4grams of SDS binds per
gram of protein.
• Mercaptoethanol assists the protein
denaturation by reducing all disulfide
bonds.
The Equipment
SDS-PAGE set
Power supply
Boiling water for preparation sample.
Gel preparation
Separation gel contents

components Amount
1.5 M Tris-HCL PH 8.8 2 ml

H2O 2.8 ml

10% SDS 80µl

10% Ammonium persulphate 100 µl


(fresh)
TEMED 20 µl

Acrylamide stock 3.2 ml


Stacking gel contents

components Amount
1.5 M Tris-HCL PH 6.8 1 ml

H2O 3 ml

10% SDS 80 µl

10% Ammonium 100 µl


persulphate (fresh)
TEMED 20 µl

Acrylamide stock 1 ml
Running buffer pH 8.4

components Amount
Tris 15 g

Glycine 72 g

SDS 5g

Made up to 1L with distilled water


Disruption buffer(sample buffer)
components Amount
20% (w/v) SDS 1 ml

1M Tris HCL pH 0.5 ml

Glycerol 1 ml

B- mercaptoethanol 0.5 ml

Bromophenol blue 0.01 g

Made up to 10 ml with distilled water

reparation
sults, all samples should be in identical, low ionic strength buffers.
of each sample with 10 μl of disruption buffer.
boiling water bath in for 2 min.
ases,brief boiling 3 min improves denaturation, but it may also cause the protein to precipi
Gels
SDS PAGE
Due to high density of binding of
SDS to proteins, the ratio
size/charge is nearly the same
for many SDS denatured
proteins. Hence proteins are
separated only by length of their
polypeptide chains (but not by
differences in charge).
Great separation. Allows
estimation of the size of
polypeptide chains
PAGE

Separate native proteins by


size –
Protein visualization on gels
• Immediately after electrophoresis proteins in the gels are
often stained by Coomassie Blue dye.Stain the gel for 1
hour, agitate it slowly on a shaker.

• Destain the gel in a destaining solution a few times until


protein bands are visualised.

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