The document outlines advanced techniques in pharmaceutical research, focusing primarily on sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) for protein separation based on molecular weight. It details the preparation of gels, sample processing, and the operational principles of SDS-PAGE, alongside additional techniques like agarose gel and starch gel electrophoresis. Various staining methods used for protein visualization and the applications of these techniques in analyzing complex protein mixtures are also discussed.

1. SDS-PAGE
and other
advanced
techniques

2. SDS PAGE AND OTHER ADVANCED TECHNIQUES
Assignment of
METHODS IN PHARMACEUTICAL RESEARCH (MPR)
PHS CC 1201
Session 2023-2024
Department of Pharmaceutical Sciences
Dr. Harisingh Gour Vishwavidyalaya,Sagar, (M.P.)
(A Central University)
Supervisors:
PROF.ASMITAGAJBHIYE
DR. UDITA AGRAWAL
MR.SHIVAM KORI
Submitted by:
ARYA OJHA
Y23254025

3. ACKNOWLEGEMENT
Throughout my assignment, I truly appreciate the help and encouragement I received from my teachers
PROF. ASMITA GAJBHIYE, DR. UDITA AGRAWAL and MR. SHIVAM KORI.

4. TABLE OF CONTENT :
1. INTRODUCTION
2. ABOUT SDS-PAGE
3. GEL PREPARATION
4. PROCEDURE
5. OTHER TECHNIQUES

5. 1. INTRODUCTION
 Sodium Dodecylsulfate Polyacrylamide Gel
Electrophoresis(SDS-PAGE) is a standard test that used to separate
proteins on basis of its molecular weight.
 Widely used in biochemistry, forensics, genetics and molecular
biology.
What is SDS?
 Negatively charged detergent sodium dodecylsulfate (SDS)
 Used to denature and linearize the proteins
 Coated the proteins with negatively charged

6. 1. Firstly, the instrument of SDS-PAGE is prepared.
GEL PREPARATION
 Casting stand
 Tall and Short Glass Plate
 Green Casting Frame
 Comb

7. 1 cm below comb a mark
is put as resolving gel is
prepared till here only.
• Leakage is checked if any.
• Leakage is checked using
Water.
• Then Resolving gel is
prepared.

8. Preparation of Resolving Gel:
 Acrylamide is used. (More concentration, Gel pore size less)
 It is 1.5M Tris with pH 8.8
 Acrylamide is inert material so when protein sample is runned it does not
interact.
 Tris is a buffer solution to maintain the pH of Gel.
 SDS- Sodium Dodecyl Sulfate used.
- breaks non-covalent bond between proteins
-coverts its tertiary and secondary structure to primary structure
-to this primary structure of protein, SDs molecules bind
 Distilled water
 APS (Ammonium Per Sulfate)
 TEMED(N,N,N’,N’ Tetramethylrthylrnr-1,2-diamine)
Role of APS and TEMED:
The acrylamide undergoes polymerization for
Gel formation.
 APS is used for generating the acrylamide
free radicals so that free radical
polymerization is initiated.
 TEMED is free radical stabiliser and is
added to promote polymerisation

9. • The solution was left
to get solidified.
• It was filled upto the
black mark shown
earlier.
• Now, Stacking gel is
prepared.
• The ingredient is same as
resolving gel, but the
concentration of
Acrylamide is changed.
Preparation of Stacking Gel:
 Acrylamide is used. (Less concentration, Gel pore size
more)
 It is 1.5M Tris with pH 6.8
 SDS
 Distilled water
 APS (Ammonium Per Sulfate)
 TEMED(N,N,N’,N’ Tetramethylrthylrnr-1,2-diamine)

10. The Stacking gel is poured from side of the comb.
It is ensured that bubble formation is not there.
The Gel is left to solidify.
Then the comb is removed because of which Well-Formation takes place.
In this well only, Sample is prepared.
Running Buffer is prepared using Tris-Glycine with pH 8.3.
Preparation Sample:
 Tris-HCl maintains pH as well as provides
Cl ions.
ᵦ - Mercaptoethanol breaks SH bond of
proteins.
 Bromophenol Blue for Sample visualisation.
 Glycerol for providing viscosity to sample
solution as sample can come out of well.

11. • Well formation in
which sample is loaded.
• Lid is closed by
taking care of anode
and cathode.
• Electric current is
passed through it.
• Running buffer is added.

12. PRINCIPLE OF SDS-PAGE:
 Separates proteins 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 Tris-HCl provides Cl- ions.
In Stacking Gel movement:
Cl-
SDS-Protein
Glycine as pH of stacking is 6.8
In Running Gel movement:
Cl-
Glycine as pH of running is 8.8 so Glycine starts becoming negatively charged
SDS-Page
More acrylamide, less gel pore size so smaller proteins moves first than larger
proteins and then separate out.

13. Visualization of Protein Bands:
• Visualize the band under UV.
• Types of Stains:
1. Coomassie Blue
2. Silver stain
APPLICATIONS:
Determine molecular weight of protein
Quantifying protein

15. 2. OTHER TECHNIQUES
TECHNIQUES
AGAROSE GEL
ELECTROPHORESIS
STARCH GEL
ELECTROPHORESIS
2-D PAGE
ELECTROPHORESIS

16. AGAROSE GEL
ELECTROPHOR
ESIS:
Commonly
used support
medium
Less expensive than
cellulose acetate
Equally good
separation
Agar is a complex acidic
polysaccharide containing
monomers of sulfated
galactose
Agarose is a sulfate
free fraction of Agar
Gel is prepared in
buffer and spread
over a microscopic
slide
A small sample of
serum or biological
fluid is applied by
cutting into the gel
with a sharp edge
The electrophoretic
rum takes about 90
minutes

17. STARCH GEL ELECTROPHORESIS:
• A suspension of granular starch should be boiled in a buffer to give a clear colloidal suspension.
• The suspension on cooling sets as a semisolid gel due to intertwining of the branched chains of amylopectin.
• To avoid swelling and shrinking petroleum jelly is used.
• ADVANTAGES:
-High resolving power and sharp zones are obtained.
-The components resolved can be recovered in reasonable yield, especially proteins.
-Can be used for analytical as well as preparative electrophoresis.
• DISADVANTAGES:
-Electro osmotic effect.
-Variation in pore size from batch to batch.

18. 2-D ELECTROPHORESIS:
• It is a powerful and widely used method for the analysis of complex
protein mixtures extracted from cells, tissues, or other biological samples.
• It is the method available which is capable of simultaneously separating
thousands of proteins.
• This technique separate proteins in two steps, according to two
independent properties:
• First-dimension is isoelectric focusing (IEF), which separates proteins
according to their isoelectric points (pI);
• Second-dimension is SDS-polyacrylamide gel electrophoresis (SDS-
PAGE), which separates proteins according to their molecular weights
(MW).
• In this way, complex mixtures consisted of thousands of different proteins
can be resolved and the relative amount of each protein can be determined.

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