Two dimensional gel electrophoresis - Principle and protocols with applications

1. TWO-DIMENSIONAL GEL ELECTROPHORESIS
(2DGE)
Shyam K U
MFSc (AAHM) student
ICAR-CIFE, Mumbai, India
INTRODUCTION
Electrophoresis is the motion of dispersed particles relative to
a fluid under the influence of a spatially uniform electric field. It is an
analytical method frequently used in molecular biology and medicine. It
is applied for the separation and characterization of proteins, nucleic acids
and subcellular-sized particles like viruses and small organelles. Its
principle is that the charged particles of a sample migrate in an applied
electrical field. If conducted in solution, samples are separated according
to their surface net charge density. Gel electrophoresis is a method for
separation and analysis of macromolecules (DNA, RNA, and proteins)
and their fragments, based on their size and charge. There are two types
of gel electrophoresis – one dimensional and two dimensional
2-D electrophoresis is a powerful and widely used method for the analysis
of complex protein mixtures extracted from cells, tissues, or other
biological samples Two-dimensional electrophoresis was first introduced
by O’Farrell and Klose in 1975.This 2DGE arises from a question that
how we can separate two proteins of having the same molecular mass
because this two protein together will give one band after the SDS-PAGE.
So we cannot distinguish these two proteins.
This technique separates proteins according to
two independent properties in two discrete steps The first-dimension step,
isoelectric focusing (IEF), separates proteins according to their isoelectric
points (pI); the second-dimension step, sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE), separates proteins
according to their molecular weights. Each spot on the resulting two-
dimensional .gel potentially corresponds to a single protein species in the

2. sample. Thousands of different proteins can thus be separated, and
information such as the protein pI, the apparent molecular weight, and the
amount of each protein can be obtained. In the original technique, the first
dimension separation was performed in carrier-ampholyte-containing
polyacrylamide gels cast in narrow tubes. carrier-ampholyte-generated
pH gradients have been replaced with immobilized pH gradients, and tube
gel replaced with gel supported by a plastic backing.
Immobiline DryStrip gels offer a marked improvement over tube gels
using carrier ampholyte–generated pH gradients. When Immobiline
DryStrip gels are used for the first-dimension separation, the resultant 2-
D spot maps provide superior results in terms of resolution and
reproducibility
PRINCIPLE
FIRST STEP:- FIRST DIMENSIONAL ISOELECTRIC
FOCUSING (IEF)
Isoelectric Focusing is an electrophoretic method that separates proteins
according to their isoelectric points (pI). Proteins are amphoteric
molecules; they carry either positive, negative, or zero net charge,
depending on the pH of their surroundings The net charge of a protein is
the sum of all the negative and positive charges of its amino acid side
chains and amino- and carboxyl-termini. The isoelectric point (pI) is the
specific pH at which the net charge of the protein is zero. Proteins are
positively charged at pH values below their pI and negatively charged at
pH values above their isoelectric point.
The presence of a pH gradient is critical to the IEF technique. In a pH
gradient and under the influence of an electric field, a protein will move
to the position in the gradient where its net charge is zero. A protein with
a net positive charge will migrate toward the cathode, becoming
progressively less positively charged as it moves through the pH gradient
until it reaches its pI. A protein with a net negative charge will migrate
toward the anode, becoming less negatively charged until it also reaches
zero net charges. If a protein should diffuse away from its pI, it

3. immediately gains charge and migrates back. This is the focusing effect
of IEF, which concentrates proteins at their pI’s and allows proteins to be
separated on the basis of very small charge differences
The resolution is determined by the slope of the pH gradient and the
electric field strength. IEF is therefore performed at high voltages
(typically in excess of 1000 V). When the proteins have reached their final
positions in the pH gradient, there is very little ionic movement in the
system, resulting in a very low final current (typically in the microamp
range). IEF of a given sample in a given electrophoresis system is
generally performed for a constant number of Volt-hours (Volt-hour [Vh]
being the integral of the volts applied over the separation time).
IEF performed under denaturing
conditions gives the highest resolution and the sharpest results. Complete
denaturation and solubilization are achieved with a mixture of urea,
detergent, and reductant, ensuring that each protein is present in only one
conformation with no aggregation, therefore minimizing intermolecular
interactions.

4. 2. SECOND STEP:- 2-DIMENSION SDS-PAGE
SDS-PAGE is an electrophoretic method for separating polypeptides
according to their molecular weights (Mr). The technique is performed in
polyacrylamide gels containing sodium dodecyl sulfate (SDS). The
intrinsic electrical charge of the sample proteins is not a factor in the
separation due to the presence of SDS in the sample and the gel. The
polyacrylamide gel is cast as the separating gel (resolving or running gel),
topped by usually the stacking gel, and secured in an electrophoresis
apparatus. But here the IPG strip is placing instead of stacking gel. The
proteins are get denatured and solubilized in the presence of 0.1% SDS
upon heating. When the SDS in solution in water, forms globular micelles
composed of 70–80 molecules with the dodecyl hydrocarbon moiety in
the core and the sulfate head groups in the hydrophilic shell. SDS and
proteins form complexes with a necklace-like structure composed of
protein-decorated micelles connected by short flexible polypeptide
segments (77). The result of the necklace structure is that large amounts
of SDS are incorporated in the SDS-protein complex in a ratio of
approximately 1.4 g SDS/g protein. SDS masks the charge of the proteins
themselves and the formed anionic complexes have a roughly constant net
negative charge per unit mass
In addition to this, by adding thiols such as Dithiothreitol
(DTT) or 2- mercaptoethanol (2-ME), reduces disulfide bonds, ensuring
that only single polypeptides are resolved. This will lead to a constant
charge to mass ratio and identical charge densities for the denatured
proteins. The constant charge to mass ratio means that the protein migrates
through the electrophoresis gel based on size – the larger the protein, the
more it interacts with the gel pores and the more slowly it migrates. Thus,
the SDS- Protein complex are migrated in the gel according to size, not
charge.

5. Polyacrylamide gels form through the copolymerization of monomeric
acrylamide to form the long chains and crosslinking of those chains by
bisacrylamide monomer (N,N’–methylene bisacrylamide). The
polymerization agent used is Ammonium Per Sulfate (APS) 10% and
N,N,N’,N’–tetramethylene diamine(TEMED) as a catalyst for the
formation of free radicles from APS. This should be added just before
casting the gel. Most proteins are resolved on polyacrylamide gels
containing from 5% to 15% acrylamide and 0.2% to 0.5% bisacrylamide
(1:37.5). The ratio will increase when the sample contains large proteins
and vice-versa
PROTOCOL
1. Sample Preparation
For eg : Prepare cell lysates with Trizol extraction. Determining
Protein Concentration of cell lysates and dilute with Buffer. For
aqueous samples, dilute a portion of the protein sample to be
analyzed 1:1(v/v) with IEF buffer
2. First dimension Separation: Isoelectric Focusing (IEF) : using
Immobilized pH Gradient (IPG) strips
Immobilized pH gradients are formed using two solutions, one
containing a relatively acidic mixture of acrylamide buffers and the
other containing a relatively basic buffer mixture. Added to
polyacrylamide gel and cast onto a plastic backing and cut into 3mm
wide strips, then dry it.
(i) Select an appropriate IPG strip size according to your sample
volume
(ii) Prepare the strip holder(s) corresponding to the IPG strip
length chosen for the experiment. Handle the ceramic strip
holder with care, as they are fragile.
(iii) Wash strip holder: Rinse each strip holder with ddH2O
followed by adding a few drops of Holder Cleaning Solution.

6. Use a toothbrush and vigorous agitation to clean the strip
holder. Rinse well with ddH2O. Thoroughly dry the strip
holders with lint-free tissue prior to use.
(iv) Prepare fresh rehydration solution
For example: thaw 945uL rehydration stock (stock in the –
80°C freezer), add 50uL of 1M DTT and 5uL of IPG buffer
solution mix well.
(v) Prepare the sample and rehydration solution mixture: add
appropriate volume of rehydration solution into the sample to
make up the total volume indicated in the following table
Table 2. Sample and rehydration solution mixture volume per IPG Strip
IPG Strip length (cm) Total volume per strip (uL)
7 125
11 200
13 250
18 340
24 450
(vi) Distribute the rehydration-sample solution mixture evenly in
the strip holders. Be sure to record the boat number with the
sample name.
(vii) Remove the protective cover from the IPG strip starting at the
positive (acidic, pointed or arrow) end. Position the IPG strip
in the re-swelling holder with the gel side down and the
positive end of the strip against the sloped end of the slot.
(viii) Overlay each IPG strip with Dry Strip Cover Fluid to minimize
evaporation and urea crystallization.
(ix) Run IPG strip on the IEF apparatus.
(x) Once the program is complete, turn off the power and remove
your samples.

7. (xi) IPG strips can either be stored in a plastic Petri dish at -80°C
(gel side facing up) or directly follow SDS-PAGE
3. Second dimension Separation: SDS-Page
(i) Make an SDS-polyacrylamide gel without the upper stack, leaving
about 50mm of the room from the top.
(ii) If your strips are stored at -80°C, remove the IPG strips from
the freezer and allow them to thaw for 10 to 20 minutes prior to
equilibration of strips
(iii) If your strips are stored at -80°C, remove the IPG strips from
the freezer and allow them to thaw for 10 to 20 minutes prior to
equilibration of strips
(iv) Wash IPG strips with SDS-PAGE running buffer (1x) before
loading to the second-dimension gel in order to get rid of the free
DTT and IAA.
(v) Apply the equilibrated IPG strips to the second-dimension gel
between the two glass plates. Make sure the IPG plastic backing is
against ONE of the glass plates.
(vi) Seal the IPG strip in place: Add about 1mL of agarose sealing
mixture over the gel strip and filter paper to completely cover them.
Allow 10 to 15 minutes to solidify.
(vii) Run the gel: Generally, the higher the voltage, the faster your
samples will run through the gel. However, at too high of a voltage
melting as well as a poor visual resolution of your gel will occur.
(viii) Stain the gel with Coomassie, Silver or SYPRO Ruby staining
to determine protein levels or with ProQ Diamond staining to detect
phosphorylated proteins
4. Visualization
REAGENTS REQUIRED
Solution Component Amount (10 mL)
Bromophenol blue 100 mg
1% Bromophenol Blue Stock solution:

8. Tris-base 60 mg
Deionized water (Milli-Q) Up to 10 mL
Rehydration Buffer:
Solution Component Amount (10 mL)
Urea 4.2 g
Thiourea 1.52 g
CHAPS 0.4 g
1 M DTT* 500 µL
100% IPG buffer (Ampholines)* 50 µL
1% Bromophenol blue 200 µL
Deionized water (Milli-Q) Up to 10 mL
*If freezing Rehydration Buffer do not add DTT or IPG buffer until ready to
use
SDS Equilibration Buffer:
Solution Component Amount (200 mL)
1 M Tris, pH 8.8 10 mL
Urea 72.07 g
100% Glycerol 60 mL
10% SDS 40 mL
1% Bromophenol Blue 400 µL
Deionized water (Milli-Q) Up to 200 mL
This is a stock solution. Prior to use DTT or iodoacetamide are added
12% SDS-Polyacrylamide Gel:
Solution Component Amount (10 mL)
Deionized water (Milli-Q) 3.3 mL
30% Acrylamide 4.0 mL
1.5 M Tris, pH 8.8 2.5 mL

9. 10% SDS 100µL
10% APS 100µL
TEMED 4µL
Agarose Sealing Mixture:
Solution Component Amount
1X TAE Buffer 50 Ml
Agarose 0.5 g
1% Bromophenol Blue 100 µL
SDS-PAGE Running Buffer (10x):
Solution Component Amount (4L)
Glycine 576g
Tris base 121.1g
SDS 40g
ddH2O top up to 4L
APPLICATIONS
A large and growing application of 2-D electrophoresis is
within the field of proteomics. The analysis involves the systematic
separation, identification, and quantitation of many proteins
simultaneously from a single sample. Two-dimensional
electrophoresis is used in this field due to its unparalleled ability to
separate thousands of proteins simultaneously. The technique is also
unique in its ability to detect post- and co-translational
modifications, which cannot be predicted from the genome
sequence. Applications of 2-D electrophoresis include proteome
analysis, cell differentiation, detection of disease markers, therapy
monitoring, drug discovery, cancer research, purity checks, and
micro-scale protein purification.