IJCAT - International Journal of Computing and Technology

Volume 1, Issue 1, February 2014

www.IJCAT.org

95

Gel Electrophoresis Technique for Protein
Identification


1
Ms. Priyanka Dudhe,
2
Ms. Ashwini Yerlekar


1, 2
Department of Computer Technology, Yeshwantrao Chavan College of Engineering
Nagpur, Maharashtra, India



Abstract - Gel electrophoresis is a wide and powerful used
methodology for the analysis of complicated proteins extracted
from tissues, cells, or different biological samples. This method
separate proteins in two steps, consistent with two freelance
properties: the first-dimension is isoelectric focusing (IEF) that
separates proteins consistent with their isoelectric points (pI);
the second-dimension is SDS-polyacrylamide gel electrophoresis
(SDS-PAGE), which separates proteins consistent with their
molecular weights (MW). During this paper there's complete
description regarding second gel electrophoresis technique.

Keywords - Gel electrophoresis, proteins, tissues, cells,
isoelectric focusing, isoelectric points, SDS- polyacrylamide
gel electrophoresis.

1. Introduction

The term electrophoresis was originally meant to refer
to the migration of charged particles in an electrical field.
The alternative term ionophoresis had been reserved for
the migration of lower molecular weight substances in
stabilized media such as gels and powders. Today, the
general term electrophoresis covers all applications
regardless of the material being studied and the medium
being used. [3]

Two-dimensional electrophoresis was first introduced by
P. H. OFarrell [1] and J. Klose [2] in 1975. In the
original technique, the first-dimension separation was
performed in carrier ampholyte-containing
polyacrylamide gels cast in narrow tubes.[4]

Two-dimensional gel electrophoresis (2-DE) methods
such as two-dimensional polyacrylamide gel
electrophoresis and two-dimensional difference gel
electrophoresis are popular techniques for protein
separation because they allow researchers to characterize
quantitative protein changes on a large scale. Thus, 2-DE
is frequently used as an initial screening procedure
whereby results obtained generate scientific ideas for

study. These technologies revolutionized the field of
proteomics and biomarker discovery in their ability to
detect protein changes either in differential expression or
modification [5]

2. Sample Preparation

Effective sample preparation could be a key for the
success of the experiment. The sample dictates the kind of
extraction technique used, and therefore the solubility,
charge, and pl of the proteome s of interest have an effect
on the tactic of solubilisation. The proteome fraction used
for 2-D action should be solubilised in a very denaturing
answer of low ionic strength. This answer cannot contain
elements that alter proteome size or charge. Sample
preparation conjointly involves no obligatory steps to
consume rife proteins, reduce the complexity of the
protein mixture, or choose a sub proteome of interest.
In order to characterize specific proteins in a very
advanced protein mixture, the proteins of interest should
be fully soluble electrophoresis conditions. Totally
different treatments and conditions are needed to
solubilise differing types of protein samples; some
proteins are naturally found in complexes with
membranes, nucleic acids, or different proteins, some
proteins type varied non-specific aggregates, and a few
proteins precipitate when removed from their traditional
surroundings. The effectiveness of solubilisation depends
on the selection of cell disruption technique, protein
concentration and dissolution technique, alternative of
detergents, and composition of the sample answer. If any
of those steps aren't optimized for a specific sample,
separations are also incomplete or distorted and data is
also lost.

To fully analyze all living thing proteome s, the cells
should be effectively discontinuous. Alternative of
disruption technique depends on whether or not the
sample is from cells, solid tissue, or different biological
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material and whether or not the analysis is targeting all
proteins or simply a specific sub-cellular fraction.

In order to attain a well-focused first-dimension
separation, sample proteins should be fully disaggregated
and absolutely solubilised. No matter whether or not the
sample could be a comparatively crude lysate or extra
sample precipitation steps are utilized, the sample
resolution should contain sure parts to make sure
complete solubilisation and denaturation before first-
dimension IEF. These continually embody carbamide and
one or a lot of detergents. Complete denaturation ensures
that every proteome is gift in just one configuration which
aggregation and building block interaction is avoided.
The lysis resolution, that contains urea and also the
zwitterionic detergent CHAPS, has been found to be
effective for solubilising a large vary of samples.
Reductant and IPG Buffer also are oftentimes accessorial
to the sample resolution to reinforce sample solubility [6].

3. First-Dimension Isoelectric Focusing

The first-dimension separation of 2-D ionophoresis is
IEF, wherever proteome s area unit separated on the idea
of variations in their pl. The pl of a proteome is that the
hydrogen ion concentration at that it carries no internet
charge, and it's a characteristic charged that's determined
by the amount and kinds of charged teams the proteome
carries [7].

IEF return till a gradual state is reached. Proteins
approach their pl values at a distinct rates however stay
comparatively mounted at those pH scale values for
extended periods. This can be in distinction to standard
activity, wherever proteins still move through the medium
till the electrical field is removed. In IEF, proteins
migrate to their steady-state positions from anyplace
within the system. IEF for 2-D activity is performed
underneath denaturing conditions in order that proteins
square measure utterly disaggregated and every one
charged teams square measure exposed to the majority
answer. Consequently, resolution is best underneath
denaturing condition. Complete denaturation and
solubilisation square measure needed to reduce
aggregation and building block interactions, therefore
making certain that every protein is gift in precisely one
configuration.[6]

4. Second-Dimension SDS-PAGE

SDS-PAGE (SDS-polyacrylamide gel electrophoresis) is
electrophoresis methodology for separating polypeptides
per their molecular weights. The technique is performed
in polyacrylamide gels containing sodium dodecyl sulfate
(SDS). The intrinsic electrical charge of the sample
proteins is not an element within the separation because
of the presence of SDS within the sample and therefore
the gel. SDS is associate degree anionic that, once in
solution in water, forms globular micelles composed of
7080 molecules with the dodecyl hydrocarb on moiety
within the core and therefore the sulfate head teams
within the hydrophilic shell. SDS and proteins form
complexes with a necklace-like structure composed of
proteome -decorated micelles connected by short versatile
peptide segments [8] When proteins square measure
treated with each SDS and a reluctant, the degree
electrophoresis separation among a polyacrylamide gel
depends mostly on the relative molecular mass of the
protein. In fact, there's associate degree around linear
relationship between the index of the relative molecular
mass and therefore the relative distance of migration of
the SDS-polypeptide advanced [6].

The most unremarkably used buffer system for second-
dimension SDS-PAGE is that the tris-glycine system
represented by Laemmli [9]. This buffer system separates
proteins at high pH scale that confers the advantage of
lowest proteome aggregation and clean separation even at
comparatively serious proteome masses. The Laemmli
buffer system has the disadvantage of a restricted gel
shelflife. Ettan DALT formed gels utilize a replacement
buffer system supported piperidinopropionamide (PPA),
which mixes long shelflife with the high separation pH
scale of the Laemmli system. Alternative buffer systems
can even be used, notably the Tris-tricine system of
Schgger and von Jagow [10] for resolution of
polypeptides within the Mr below ten 000. ExcelGel
formed gels for second-dimension SDS-PAGE on the
Multiphor II flatbed system utilize a distinct Tris-tricine
buffer system [6].

5. Detection

In 2-D electrophoresis, proteins in gels square measure
most typically visualized total protein stains. Choice of
the foremost acceptable stain involves thought of the stain
characteristics, limitations with relation to the sensitivity
of detection and therefore the varieties of proteome s it
stains best, downstream application, and therefore the
form of imaging instrumentality accessible. To be used in
proteomics applications. Stains ought to be compatible
with high throughput protocols and downstream analysis,
as well as digestion and mass spectrometry. It is
additionally attainable to label proteome samples
preparation and before IEF with fluorescent dyes like the
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CyDye DIGE fluors. At the time of writing, three dyes
with spectrally totally different excitation and emission
wavelengths were accessible, permitting labelling of up to
a few totally different samples and their separation during
a single 2-D gel. The dyes square measure matching for
size and charge to get migration of otherwise tagged
samples square measure mixed along before they're
applied on the gel of the primary dimension. Once
separation, the gels square measure scanned with visible
light imagers at the various wavelengths.[11]

SYPRO Ruby was one in every of the first Fluorescent
proteome gel stains, and it is a mix of high sensitivity and
wide dynamic vary that can't be achieved with ancient
Coomassie blue or silver stains. SYPRO Ruby has two
distinguished absorbance peaks. Detection sensitivity in
SYPRO Ruby stained gels will be low. SYPRO Ruby
stains most categories of proteinswith very little proteome
to protein variability. The principle advantage of SYPRO
Ruby is its skilfulness with relation to imaging needs. It
is, however, time overwhelming to use and doesn't turn
out the high-quality mass qualitative analysis knowledge
generated with alternative fluorescent stains. [12]

No stain interacts with all proteome s during a gel in
precise proportion to their mass, and every one stains act
otherwise with totally different proteome s [13]. The sole
observation that appears to use for many stains is that they
act best with proteins with a high basic organic compound
content [7].

6. Image Acquisition and Analysis

6.1 Image Acquisition

This prepares every raw acquisition for succeeding
comparative analysis. Once scanning, the pictures area
unit pre-processed by cropping, noise suppression, and
background subtraction. a picture capture device is
needed, that there area unit 3 main categories:

Flatbed scanner: This automatically sweeps a customary
charge-coupled device (CCD) underneath the gel and
might be wont to get 1216 bits of greyscale or colour
measure from actinic ray stains.

CCD camera: Since the detector is fastened, its bigger
size and cooling provides a dramatic improvement in
noise and so dynamic vary (up to 104). Totally different
filters and transillumination choices enable a large vary of
stains to be imaged, together with actinic ray, fluorescent,
reverse, luminescence, and hot signals.
Laser scanner: Photomultiplier detectors area unit
combined with optical device light-weight Associate in
Nursingd optical or mechanical scanning to pass an
excitation beam over every target pixel [37].

6.2 Image Warping

Correction of point spot variations by image warp. 2-D
ionophoresis leads to spot patterns with variations within
the spot positions between gels. Therefore, gel pictures
area unit positionally corrected by a mix of world and
native image transforms (image warping). The knowledge
concerning variations in spot positions that was gained
during this step is reused later for image fusion and for
the transfer of the agreement spot pattern.

6.3 Image Fusion

Image fusion and proteome maps condense the image info
of the complete experiment into one fusion image,
additionally known as a proteome map. The proteome
map contains the knowledge of all proteome spots ever
detected within the experiment [15].

6.4 Spot Detection and Edition

Spot detection is performed on the proteome map. As a
result, a agreement spot pattern is generated, that is valid
for all gels within the experiment. It describes the position
and also the general form of all proteome spots from the
experiment.

6.5 Application of Agreement Spot Pattern

For spot division and building expression profiles, the
agreement spot pattern is applied to any or all gel pictures
of the experiment. The image transformation assures that
each one spots of the agreement pattern attain their
correct position. A remodelling step makes certain that
the preset spot boundaries from the agreement area unit
tailored to the important grey levels discovered on the
target image. All boundaries of the agreement pattern will
be found on each gel.

6.6 Extracting Expression Profile

Expression profile analysis identifies fascinating spots
which is able to be marked for any analysis, proteome
identification, and interpretation [14].
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7. Existing Software

The commercially accessible code performs the analysis
work flow in 2 other ways. The classical package
condensed the knowledge onto spots. The spot detection is
performed before matching and expression profile
extraction. The second image analysis code cluster relies
on the complete image info. These packages apply a
warp procedure to get rid of running variations between
gels, and also the spot detection and proteome expression
profiles extraction occurred in a separated and
freelance step. [16]

The emphasis during this analysis code has been on
reducing the perspicacity of the image analysis. the actual
fact that the alignment step is performed before the spot
detection facilitates coincident spot detection on all gel
pictures in Associate in Nursing experiment and also the
ensuing spot boundaries area unit identical on all gel
pictures.[17]

7.1 Dimension

Dimension is revolutionary computer code which will
analyse a typical 2D gel image speedily. It options novel
algorithms for background subtraction, noise filtering,
and precise alignment, spot detection, fast matching and
reduced image written material time. Victimisation its
powerful spot detection algorithmic program, Dimension
instantly locates and analyses macromolecule spots. With
Dimension, the whole analysis method from background
correction to identify matching results and news takes
minutes, creating this the quickest 2D gel analysis
package presently on the market.

7.2 Delta2D and Pangenesis Same Spots

We tend to should initial manually determine many spots
which will be matched unambiguously in each gel within
the set. The spots ought to be opened up equally over the
gels surface otherwise some regions are going to be
aligned too poorly currently to be corrected later. The
computer code can then (or when each landmark)
mechanically generate a swimmingly interpolated warp
that aligns these landmarks and estimates the mediator
alignment between them.

If on the market, an extra automatic section is initiated
that adjusts the mediator alignment to higher match the
remaining spots. These matches is iteratively accepted or
changed by the user and therefore the algorithmic
program rerun. Finally, alignments should be completed
by hand and a spot mask applied to the reference gel of
every set.

Several analysis teams have developed package systems,
they're given below. But no one has developed an entire
package freely out there and platform freelance able to
perform all the steps of a 2D-GE gel analysis experiment.
[18].

7.3 PDQuest

PDQuest could be a computer code package for imaging,
analyzing, and databasing raw 2-D activity gels. The
computer code runs during a Windows or Macintosh
surroundings and contains a graphical interface with
normal pull-down menus, toolbars, and keyboard
commands. PDQuest will acquire pictures of gels
victimisation any of many Bio-Rad imaging systems. a
picture of a gel is captured victimisation the controls
within the imaging device window and displayed on your
display screen. The scanned gel will then be cropped,
rotated, etc. victimisation the image written material
controls.

7.4 Melanie

Developed by the Swiss Institute of Bioinformatics (SIB).
Identification, quantification and matching of gels for
each single and large-scale 2D comparison studies.
Automatic likewise as interactive gel analysis.
Comprehensive annotation prospects, advanced applied
math and classification capabilities, and a flexible
question engine and news functions. To envision gels and
connected information obtained through the employment
of the complete version of Melanie by colleagues.
Restricted information analysis on gels that have already
been matched with the complete Melanie computer code
package (e.g., spots, pairs and teams cannot be altered or
modified).

7.5 Flicker

Flicker is Associate in Nursing ASCII text file complete
worm for visually comparison second gel pictures. Two-
dimensional polyacrylamide gel electrophoresis (2D-
PAGE) gels are usually tough to match as a result of
rubber-sheet distortions. Flicker permits you to visually
compare your gel pictures against one another or against
those found in web databases. Several revealed web gels
have a set of spots known which can build them helpful to
match together with your gels. a number of these web gels
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are active maps that you simply will click on a spot to
inquire of its identity. We will be able to draw reputed
conclusions on the identification of some spots in your
gels that visually seem to be an equivalent spots as in
reference gels. The sparkle program integrates these
varied must assist you try and build reputed spot
identifications.

8. Conclusions

Gel electrophoresis is a wide and powerful used
methodology for the analysis of complicated proteins
extracted from tissues, cells, or different biological
samples. This system separate proteins in two steps: the
first-dimension is isoelectric focusing (IEF), that separates
proteins in keeping with their isoelectric points (pI); the
second-dimension is SDS-polyacrylamide gel
electrophoresis (SDS-PAGE), that separates proteins in
keeping with their molecular weights (MW). During this
means, complicated mixtures consisted of thousands of
various proteins are often resolved and also the relative
quantity of every protein are often determined. The
procedure involves inserting the sample in gel with a pH
gradient, and applying a possible distinction across it.
Within the electrical field, the protein migrates a
protracted the pH gradient, till it carries no overall
charge. This location of the protein within the gel
constitutes the apparent pI of the protein. The IEF is that
the most important step of the 2-D electrophoresis
process. The proteins should be solubilize while not
charged detergents, sometimes in high focused organic
compound answer, reducing agents and chaotrophs. To
get top quality knowledge it's essential to attain low ionic
strength conditions before the IEF itself. Since differing
types of samples take issue in their particle content, it's
necessary to regulate the IEF buffer and also the electrical
profile to every style of sample. The separation within the
second dimension by molecular size is performed in block
SDS- PAGE. Twelve parallel gels are often separated in
an exceedingly fastened temperature to attenuate the
separation variations between individual gels.

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