Separation and identification of proteins in a sample by displacement in 2 dimensions oriented at right-angle to one another.

1. 2D GEL
ELECTROPHORESI
S
SAKSHI JAIN, MS PHARMACOLOGY

2. GEL ELECTROPHORESIS
Electrophoretic separation is based on the migration of unbalanced charged
molecules in an electric field and is the most frequently used dispensation
method in the study of proteins and nucleic acids.
The main premise of electrophoretic separation is application of an electric
field that forces molecules to move through gel pores, separating them
based on their MW and total particle charge.
Large-molecular weight molecules are slowed down on the basis of gel pore
size, more specifically, larger-molecular-weight molecules are “trapped” in
regions of the gel with a higher percent concentration.
INTRODUCTION

4. USE OF GEL ELECTROPHORESIS
It is a technique used for the separation of
Deoxyribonucleic acid, Ribonucleic acid or
protein molecules according to their size and
electrical charge using an electric current
applied to a gel matrix.
Gel is a cross linked polymer whose
composition and porosity is chosen based
on the specific weight and porosity of the
target molecules.
Types of Gel: ▪ Agarose gel. ▪
Polyacrylamide gel.
Gel electrophoresis can be conducted
in either a horizontal or vertical
orientation. Horizontal gels are
typically composed of an agarose
matrix, while vertical gels are
generally composed of an acrylamide
matrix. Pore sizes of these gels depend
on the concentration of chemical
components: agarose gel pores (100 to
500 nm diameter) are larger and less
uniform compared to that of
acrylamide gel pores (10 to 200 nm in
diameter). Comparatively, DNA and
RNA molecules are larger than a linear
strand of protein, which are often
denatured prior to, or during this
process, making them easier to
analyze. Thus, DNA and RNA
molecules are more often run on
agarose gels (horizontally), while
proteins are run on acrylamide gels
(vertically).

5. LIMITATIONS OF 1D ELECTROPHORESIS
Electrophoresis in a single dimension is useful
for separation of few proteins simultaneously
but large number of proteins can not be
separated with good resolution
Complex mixtures e.g. serum, cell lysate can’t
be separated
Need technique to provide better resolution at
proteome level

6. 2D GEL ELECTROPHORESIS
Separation and identification of proteins in a sample by displacement in 2 dimensions
oriented at right-angle to one another.
First dimension: Separates proteins on pH gradient based on isoelectric point (pI) using
isoelectric focusing
Second dimension: Following IEF, proteins are resolved according to their molecular
weight using SDS-PAGE

7. Remember…
Safety First!
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9. WORK FLOW OF 2D ELECTROPHORESIS
1. Isoelectric focusing (first dimension)
2. Equilibration of IPG strips
3. SDS-PAGE (second dimension)
4. Staining – gel visualization
5. Image analysis
6. Spot picking
7. Enzymatic digestion
8. MS analysis

10. ISOELECTRIC FOCCUSING
• Protein separation according to isoelectric point
• Proteins introduced into immobilized pH gradient
• Electric field is applied in which protein migrates according
to its charge
• Protein reaches Isoelectric point (pI)
• pH = pI protein does not move in electric field owing to the
lack of charge

11. • Rehydrate IPG strips overnight in a reswelling tray at
RT using solution containing the extracted protein in
buffer (rehydration/IPG buffer)
• Passive rehydration – no voltage applied
• Active rehydration – apply low voltage
• Overlay mineral oil on rehydrated strips
• IPG strips different pH ranges (e.g. pH4-7, 3-10 etc.)
• IPG strips length are between 7-24 cm
• IEF units are capable of accommodating IPG strips of
different length (7-24 cm)
• Large gels are recommended to resolve spots better
• However, handling large gels is tedious

13. SDS PAGE
• Equilibrating IPG strips after IEF .
• Applying IPG strips to the second dimensional SDS PAGE.
• Performing SDS PAGE
REMOVE THE IPG STRIPS FROM THE TRAY
PLACE THE IPG STRIP FACING UP IN THE
EQUILIBRATION BUFFER

14. IPG strip is placed on top of the pre-cast SDS-PAGE gel and electric current apply

15. Next step
• Separation on basis of molecular weight not
isoelectric point
• Requires modest voltage
• Requires a shorter period of time
• Presence of SDS is critical to disrupting
structure and making mobility
• Degree of resolution determined by percentage
of acrylamide and electric field strength

17. Detection

20. Reference
• A. Drabik and A. Bodzon-Kułakowska ; J. Silberring ; GEL ELECTROPHORESIS;
Proteomic Profiling and Analytical Chemistry; Proteomic Profiling and Analytical
Chemistry. http://dx.doi.org/10.1016/B978-0-444-63688-1.00007-0 2016 Elsevier
• Sameh Magdeldin , Shymaa Enany , Yutaka Yoshida , Bo Xu , Ying Zhang , Zam Zureena
, Ilambarthi Lokamani , Eishin Yaoita and Tadashi Yamamoto; Basics and recent advances
of two dimensional- polyacrylamide gel electrophoresis; Magdeldin et al. Clinical
Proteomics 2014