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1. Proteomics :2D gel electrophoresis and
in-gel digestion

2. Flow of presentation
 Proteomics
 Proteomics applications
 Gel electrophoresis
 Types
 Factors Affecting gel Electrophoresis
 Gel Electrophoresis components
 Buffers
 Agarose gel electrophoresis
 Procedure of Agarose gel electrophoresis
 PAGE
 Difference
 SDS-PAGE
 Visualization
 2 D gel electrophoresis
 In gel digestion

3. Proteomics
 Proteins are the most complex of all biological molecules in terms of their structures and
functions.
 The total number of proteins expressed in a cell at a particular time is known as a proteome.
 The status of the cell can be assessed by its proteome, for example, the proteome of a healthy
cell is different from that of a diseased cell.
 Proteomics is the study of an entire complement of proteins expressed in a particular cell at a
particular time. It is a unique means of characterizing proteins that are expressed in a cell or
tissue at any given time-point and of identifying any modifications that they may undergo. Thus,
it is a powerful technology that can detect and identify the changes of the structure and
function of proteins in response to intra- and extracellular environmental signals or disease
states.

4. Proteomics
Flow chart for
any Proteomic
study

5. Proteomics in drug discovery

6. Proteomics applications
1. Molecular weight determination.
2. Isoelectric point determination.
3. Amino acid sequence.
4. Quantification of proteins.
5. Post-translational modification.
6. Peptide mapping.
7. Comparative proteomics.
8. Structure determination.
9. Biomarker identification.
10. Drug–target identification

7. Gel electrophoresis
Principle: When we place any charged molecules in
an electric field, they move toward the positive or
negative pole according to the charge they are
having.
The velocity of migration ‘v’ of molecule is
v = Eq / ƒ where
E is the Applied electric field
q is the net electric charge on molecule and
f is frictional coefficient
Father of Electrophoresis
Arne Wilhelm Kaurin Tiselius
Uppsala University, Uppsala, Sweden
The Nobel Prize in Chemistry 1948
For his research on electrophoresis
and adsorption analysis, especially for
his discoveries concerning the complex
nature of the serum proteins.

8. Factors Affecting gel Electrophoresis
Electrophoretic velocity depends on
• How much charge the particles have.
Increase charge = faster speed
• What is the molecular weight.
High molecular wt.= slow
Increase size = slow
• pH of solution
Determine net charge and direction of
movement
• Electric field
Increase=fast
• Solution viscosity
Viscous= slow
• Temperature
Increase=fast (denaturation)

9. Gel Electrophoresis components

10. Buffers

11. Gel
electrophoresis
Agarose PAGE 2D-E
Native -PAGE SDS-PAGE
Types

12. Agarose gel elecrophoresis
• Agarose is a polysaccharide extracted from seaweed (Gelidium) and contains mainly agarobiose subunits. These
gels are not toxic like acrylamide gels .
• During solidification, agarose form a network of polymers and its pore sizes can be determined by its
concentration
• It is usually used at concentrations of 0.5 to 2%. The lower the concentration of agarose, the faster the DNA
fragments migrate.
• Stiffer gel means the agarose concentration is higher
• By varying the concentration of agarose, 500 to 4000 base pairs of DNA can be separated.
Staining: Ethidium Bromide
Intercalation between base pairs of nucleic acids results in very strong binding. When EtBr is exposed to uv light,
electrons in the aromatic ring of the ethidium molecule get activated, which releases energy in the form of light.
There are other stains SYBR Gold, SYBR green, Crystal Violet and Methyl Blue.
Stock prepared : 10mg/ml Working concentration : 0.5 µg/mL.

13. Gel acts as molecular seive
concentration
s
Agarose gel elecrophoresis

14. Procedure of Agarose gel elecrophoresis
To prepare gel, Agarose powder is mixed with
electrophoresis buffer, and heated in a microwave
oven to melt it then ethidium bromide is added.
Cooled.
Then it is poured into a casting tray containing a
sample comb and allowed to solidify at room
temperature.
Gel is inserted horizontally into the electrophoresis
chamber then add running buffer. Load samples.
Run gel at constant voltage until band separation
occurs
Observe the separated DNA bands in a UV chamber

15. PAGE
• In this Agarose gel is replaced by polyacrylamide
gel.
• Polyacrylamide gels are based on the free radical
polymerization principle of acrylamide and cross-
linking N,N-methylene-bis-acrylamide.
• It is especially used for the electrophoretic
separation of small or medium sized (up to about
1 X106
Da) proteins.
• Ammonium Persulfate (APS) is an oxidizing agent
which is used in combination with N, N, N’, N’-
tetramethylene diamine (TEMED) which acts
as a catalyst to catalyze the polymerization of
acrylamide and bisacrylamide. Ammonium
persulfate forms free radicals when it is dissolved
in water and it initiate polymerization of
acrylamide solutions.

16. Difference
Agarose Gel
• Horizontal
• Low resolution compared to
PAGE
• They have large pore sizes and
are used for separating larger DNA
molecules (RFLP Analysis) or RNA
separation.
• Also used to separate large
proteins and protein complexes.
Polyacrylamide (PAGE)
• Vertical
• Used to obtain high resolution
separations.
• Used for the separation of smaller
DNA molecules (STR analysis and
DNA sequence analysis.
• They have small pore size gel, is
used to separate most proteins and
small nucleotides.

17. SDS-PAGE
 SDS PAGE ,also known as Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis is a technique used for
separating the proteins based on their molecular weight.
Principle : A charged molecule migrates to the electrode with the
opposite sign when placed in an electric field. The separation will take
place as the mobility of the charged species. The tiny molecules tend to
move faster due to their less resistance at the time of electrophoresis.
The rate of migration influences the structure and the charge of the
protein.
Sodium dodecyl sulphate help to eradicate the influence of structure
and charge of the proteins, and the proteins are separated based on
the length of the polypeptide chain.
SDS is a detergent that break the disulphide bonds of proteins
disrupting the tertiary structure of proteins.

18. Identification and separation of
protein
SDS-PAGE Procedure

19. SDS-PAGE Procedure cont…

20. Visualization
Stains such as Coomassie Brilliant Blue, silver, and Deep Purple can be employed to visualize the
proteins.
Gel staining: Coomassie blue
Chemical reagents required: Brilliant Blue R-250 (BBR), Sterile distilled water
Fixing solution: It is prepared by mixing different ratios of methanol(50), acetic acid(10) and
water(40).
Destaining solution: It is prepared by mixing different ratios of methano(45)l, acetic acid(10) and
water(45).
Coomassie concentrated stain solution: It has 12.0 g BBR to which 300 mL Methanol is added
then acetic acid (60ml) is added. After mixing all components it is stirred properly.
Coomassie Working solution: To this 500 mL Methanol we add 30 ml of Coomassie stain solution
and 400ml of water plus 100 ml of acetic acid. After mixing it is filter sterilized using a syringe filter.

21. 2 D gel electrophoresis
It is a type of gel electrophoresis where proteins are separated in two
dimensions. It is the only method which is available and is capable of
simultaneously separating thousands of proteins.
Principle
• Proteins are separated on the basis of two properties.
• Firstly they are separated on the basis of their net charge. After that,
PAGE separates the proteins on the basis of their mass.

22. Isoelectric Point
• There is a pH at which there is no net charge on a protein and this point is
called isoelectric point (pI).
• A protein has a net negative charge above its isoelectric point, and it migrates
toward the anode in an electrical field. The protein is positive below its
isoelectric point, and it migrates toward the cathode.
• Procedure of 2D gel electrophoresis involve two steps:
1. Isoelectric focusing and
2. SDS-PAGE
2 D gel electrophoresis

23. Isoelectric Focusing
• When proteins are separated by isoelectric points it is
called isoelectric focusing (IEF). Therefore, a gradient
of pH is applied to a gel and an electric potential is
applied across the gel.
• proteins will have charge at all pH values other than
their isoelectric point.
• Proteins move towards the negative end of the gel If
they are positively charged and if they are negatively
charged they will move to the positive end of the gel.
• In the first dimension proteins will move along the gel
and will accumulate at their isoelectric point.
First Dimension Electrophoresis

24. • The focusing is carried out on an equipment which supplies gradient electric current to the IPG strips.
• IPG strip has two sides, a base of plastic and the another side which have gel on it.
• After this, IPG strip is then placed into the lane in a way that the surface with gel faces downwards and is in direct
contact with the solution around it.
• Commercially available Immobilized pH Gradient (IPG)
strips are used for focusing.
• IPG strips are solid surfaces with coat of dehydrated
polyacrylamide.
• These strips are available in a range of pH gradients such
as 5-7, 3-8, 6-12 etc.
• Rehydration of strips is done with rehydration buffer
before protein loading. For rehydration of IPG strips
these IPG strips are kept in rehydration solution for 10-15
hours.
First Dimension Electrophoresis cont..

25. • This is left on the working bench for 15 hours.
• After 15 hours the IPG strips are removed from the rehydration
solution carefully using forceps
• This IPG strip is then placed in the fresh tray, with the gel side
facing upwards.
Isoelectric Focusing procedure
• The strips are put in isoelectric focusing unit.
• The voltage gradients and time intervals depends on the type
of strip that we are using to run the samples.
• The separation can be monitored directly
After focusing the strips are taken out and can be used in next
step.
First Dimension Electrophoresis Cont…

26. Equilibriation of Strips
We need to equilibrate the strips before separating them in second dimension.
• Prepare equilibration buffer I(DTT) and II(Iodoacetamide)
• Place IPG strips gel side up and add 4ml of equilibration buffer –I ,place the centrifugal tube on
shaker for 20min.
• Carefully take out the strip and place in the equilibration buffer –II for 20 min .
• Take out IPG – strips and rinse with water.
First Dimension Electrophoresis Cont…

27. Second Dimension Electrophoresis
• This separates proteins by
their sizes ( molecular).
• Normal SDS-PAGE is done
in this stage
• Stacking is replaced by IPG
strips and normal running
gel separates molecules.

28. In Gel Digestion
The in-gel digestion step is a part of the sample preparation for the mass
spectrometric identification of proteins in course of proteomic analysis. The
method was introduced in 1992 by Rosenfeld.
The basic steps are
1. Excision of spot of interest
2. Removal of staining agent
3. Reduction & alkylation
4. Tryptic digestion
5. Extraction of digested proteins:

29. Materials Required
Chemicals:
Ammonium bicarbonate (NH4HCO3) [MW: 79.06]
Dithiothreitol (DTT) [MW: 154.24]
Iodoacetoamide (IAA) [MW: 185]
Trypsine (Proteomics/ sequencing grade)
Acetonitrile (ACN) and Trifluro Acetic Acid (TFA)
Solutions:
Stain removal solution: 1:1 (v/v) ACN: 100mM
NH4HCO3. Dissolve 79.06 mg Ammonium
bicarbonate (NH4HCO3) in 10mL MilliQ water and
add 1mL of ACN to 1mL of this solution.
Dehydration solution: 2:1 mixture of ACN and
50mM ammonium bicarbonate buffer.
Reduction solution: 10mM DTT in 100mM ammonium
bicarbonate buffer. Dissolve 7.7mg DTT in 5mL of 100mM
ammonium bicarbonate buffer.
Alkylation solution: 55mM IAA in 100mM ammonium
bicarbonate buffer. Dissolve 10.16mg IAA in 1mL of
100mM ammonium bicarbonate buffer.
Trypsin solution: Dissolve 20µg lyophilized trypsin
powder in 100µL of 1mM HCl solution. Mix properly and
add 900µL of 40mM ammonium bicarbonate solution made
in 9% ACN. Store at -20O
C freezer. The concentration of
this stock solution is 20µg /mL. Store this stock solution in
different aliquots to avoid multiple freeze thaw cycles.
Extraction solution: 0.1% TFA in 60% ACN solution.

30. The spots of interest are excised for further processing. Spot excision can be performed both in automated
(using spot pickers) and manual way. In case of traditional manual approach, the selected spots are excised
using sterile scalpel under sterile dust-free environment and kept in sterile microcentifuge tubes.
1. Excision of spot of interest

31. 2. Removal of staining agent
Coomassie Brilliant Blue is widely used for staining in 1D and 2-DE PAGE for
visualization of proteins. Stain removal is essential prior to mass spectrometric
analysis. Immediately after spot picking, excised gels are washed with a mixture of
bicarbonate buffer and acetonitrile for removal of the staining agent. ACN reduces
the hydrophobic interaction between protein and the stain, while the ionic solution
decreases the ionic interaction between negatively charged dye and positively
charged protein.

32. 3. Reduction & alkylation
Treatment of the protein residues with DTT breaks the disulfide bonds completely. However, reformation of disulfide
bonds may occur and to prevent that IAA, an alkylating agent, is used. It adds acetoamide group to the sulfahydral
group and prevents the disulfide bond formation. The chemical reactions for the same are given below:

33. 4. Tryptic digestion
Prior to MS identification, proteins are digested to generate peptides. Several proteolytic enzymes are available.
Chymotrypsin, trypsin, pepsin are some of the enzymes used for proteolysis. CNBr is one of the chemical agents
that cleave the peptide bond at Methionine residue. Trypsin is most widely used proteolytic enzyme used for
protein digestion prior to MS analysis. It breaks the peptide bond at carboxyl terminal of basic amino acids such
as arginine and lysine.
Tryptic digestion of protein residues; trypsin recognizes the lysine and arginine resides and cleaves
to generate smaller peptide fragments.

34. 5. Extraction of digested proteins
After overnight incubation, peptides generated through proteolytic
digestion are extracted using extraction buffer containing 0.1%
FA/TFA in 50% ACN solution.
Efficient extraction process is essential to ensure the release of
peptides from gel-matrix to solution, which is further subjected to
mass spectrometric analysis.

35. Extraction of Proteins from Gels
• It is necessary to elute and recover proteins separated by gel electrophoresis for
further analysis.
• To recover a specific protein of interest from a gel, it has to be liberated out of
the gel matrix that imprisons them. In principle, one has to just simply reverse
the way these molecules entered the gel.
• Following methods are used for extraction of proteins from gels.
1. Diffusion Mediated Elution of Proteins from Gels
2. Protein Recovery by Dissolving Gels
3. Using Electroelution Devices

36. Locate a band on gel by rinsing
the gel with cold ddH2O and
staining for 5 min with ice-cold
0.25 M KCl and 1 mM DTT. Rinse
with ddH2O and destain for 10–
60 min with cold ddH2O and 1
mM DTT
Crushing the gel slice with a
small Teflon pestle in water or an
elution buffer containing 0.1%
SDS. Incubating the crushed gel
fragments for 4hr (36kDa
protein) or 16-24 hr (150 kDa
protein) on a rotator
The mixture is centrifuged at
maximum speed for 2 min to
pellet the crumbled gel. The
supernatant (the protein eluate)
is then transferred into a clean
microfuge tube.
Four volumes of cold acetone
(20℃) added to the protein and
the sample allowed to
precipitate for 30 min in a dry
ice–ethanol bath (Acetone
mediated precipitation)
Centrifuge at maximum speed in
a microfuge for 5 min. The
supernatant containing SDS is
removed and discarded and
acetone ppt is collected.
The ppt is solubilized and
denatured in 6M-Guanidine
hydrochloride
Diffusion Mediated Elution of Proteins from Gels

37. Protein Recovery by Dissolving Gels
• The easiest way to elute proteins from a gel matrix would be to dissolve this matrix.
• Very harsh conditions are required to recover proteins from acrylamide gels
containing the standard cross-linkerN,N-methylene bisacrylamide.
• Typically, a gel slice is incubated in 30% hydrogen peroxide at 50°C for this purpose.
This results in irreversible damage to all proteins. Other cross-linkers, such as N,N-
diallyltartardiamide (gel slice that has this cross-linker needs to be dissolved with 2%
periodic acid), N,N -(1,2-dihydroxyethylene) bisacrylamide (protein recovery requires
harsh conditions), and ethylene diacrylate (gel slice needs to be dissolved with 1 M
sodium hydroxide) have been used.

38. Electroelution Devices
• Proteins can be eluted by applying an electric field to the gel section and trapping the eluted
protein in an appropriate membrane bounded apparatus.
• This method is increasingly popular owing to the excellent recovery and reproducibility
without using any expensive apparatus.
Types
 Vertical-type eluter
 Horizontal-type electroeluter
 Bridge-type electroeluters
 Reversed electrophoresis using a discontinuous conductivity gradient
 Elution by employing a steady-state stacking buffer system
 Continuous elution devices

39. • A column for classical cylinder gels is modified.
• A dialysis membrane serves to seal the column and a support is
required to place the gel.
• After filling the column with elution buffer, proteins can leave the gel
by the electric field and migrate through the fluid until the
membrane retains them.
• Depending on the column diameter, different amounts of gel can be
handled easily and reliably.
• If a layer of hydroxylapatite captures the protein, the membrane can
be omitted. bound proteins elute by adding phosphate buffer or a
change in pH.
Vertical-type eluter

40. • A flat-bed electrophoresis tank and a special column are
needed to perform electroelution in horizontal direction. Both
ends of the column are sealed with semipermeable
membranes to constitute the elution chamber.
• This chamber is divided furthermore by a large pore membrane
(2 mm) into two compartments. In the larger division, the gel
pieces are placed. After applying the electric field, proteins
migrate into the smaller division and are retained by an
additional small pore membrane (Mr 5000). In general, air
bubbles are less problematic compared to the vertical-type
eluter.
Horizontal-type electroeluter

41. • It is especially for protein complexes, a construction with two
separate chambers for the gel. The main device is an overpass
vessel (bridge or H-shaped) . This device has two arms
connected by the bridge.
• Proteins migrate electrophoretically out of one arm, pass the
bridge and are captured in the second arm. As means for
concentrating samples, the vessel possesses a bigger arm
containing the gel material and a smaller arm for collecting the
electroeluate. Both arms are sealed by dialysis membranes.
• As stated for the vertical type elutor, the entrapment of air
bubbles can also be a problem while inserting the vessel into
the respective electrophoresis tank.
Bridge-type electroeluters

42. • In this, a standard slab gel electrophoresis device is used for electroelution.
The possibility is to cast a support gel and place the gel pieces of interest on
this support. A small volume of buffer is filled in and the chamber is sealed
by a dialysis membrane. In this case, electroelution is performed in opposite
direction to the preceding electrophoresis.
• A low-conductivity layer, preferably containing glycerol, surrounds the gel
pieces. A second solution with high-conductivity is layered upon the first one
is placed.
• Proteins in the low-conductivity zone migrate rapidly out of the gel. When
reaching the upper zone, the migration slows down and stops because now
the highly concentrated surrounding ions predominate as electrolytes. The
advantage compared to other methods is the concentration of proteins
between two solution layers without need for membranes.
Reversed electrophoresis using a discontinuous conductivity
gradient

43. • In this also the standard electrophoresis equipment can be
applied for electroelution. For this purpose, gel pieces have to
be embedded in a new stacking gel overlaid with a glycerol
layer. Therefore, proteins leaving the gel pieces are
concentrated and subsequently released into the glycerol
solution.
• An electrophoretic column setup can be applied under steady-
state stacking conditions in combination with a stabilizing
medium like Sephadex G-25 or cellulose. During ITP, an exactly
balanced buffer system is of utmost importance and determines
the success of the procedure.
Elution by employing a steady-state stacking buffer system

44. • Normally electrophoresis is stopped, before the smallest proteins
run out of the gel. Therefore, it suggests itself to continue
electrophoresis beyond this point, so that molecules leave the gel.
Subsequently, a continuous flow of buffer absorbs the eluate .
• The proteins(P1 and P2) are electrophoretically separated on the
polyacrylamide gel which is supported by the grid (G). The
separated proteins migrate into the buffer stream (indicated by
open arrow) running between the grid and semipermeable
membrane (M).
• The eluted protein that is entrapped with the buffer flow (B) is
delivered to the fraction collector via the monitoring detector (D).
Continuous elution devices

45. Dye
Sensitivi
ty
Typical
protocol
time Detection
Compatibility with downstream
applications Advantages
Coomassie
staining
5-25 ng 10-135
min
Visual Mass spectrometry (MS) and
sequencing compatible, western
blotting.
•Quick, and simple staining
protocols
•Reversible: with no permanent
chemical modification
Silver
staining
0.25-0.5
ng
30-120
min
Visual Certain formulations are MS
compatible
Lowest detection limits not
requiring specialized equipment
Fluorescent
stains
0.25-0.5
ng
60 min UV or blue/green-
light
transilluminators or
imaging instruments
with appropriate
filters
Most stains are MS compatible,
western blotting
Broad linear dynamic range with
low detection limits
Zinc stains 0.25-0.5
ng
15 min Visual MS compatible, western blotting No chemical modification of
proteins; bands are visible because
the background is stained (not the
bands)
Important Stains

46. Dye Sensitivity
Typical
protocol
time Mode of action
Compatibility with
downstream
applications Advantages
Periodate-acid-
Schiff (PAS)
reagent method
0.6-160 ng
glycoprotein
/ band
2 hr 40 min Interactions with
periodate-oxidized
carbohydrate groups at
glycosylation sites
In-gel (1D or 2D), in-blot
detection
(nitrocellulose)
No specialized equipment
required for visualization
Pro-Q Diamond
Phosphoprotein
Gel Stain
1–16 ng
phosphopro
tein / band
4–5 hr Detects phosphate groups
attached to tyrosine,
serine, or threonine
residues
In-gel (1D or 2D), mass
spectrometry
Selective staining of
phosphoproteins
Important Stains

47. 1. Characterization of proteins
2. Posttranslational modification (PTM)
3. Protein – protein interactions
4. Metabolic engineering
5. Characterization of mutant proteins
6. Microbial systematics and epidemiology
7. Evaluation of proteins involved in the toxic response & Vaccine development
Applications of 2D-E

48. 1. Characterization of proteins
• 2-DE polyacrylamide gel electrophoresis is regarded as a powerful technique, because it can be used to
separate and resolve complex protein mixtures into thousands of individual components.
• Depending on the gel size, 2-D PAGE is capable of resolving more than 5000 proteins simultaneously and can
detect less than 1 ng of protein per spot.
2. Posttranslational modification (PTM)
• There are possible modifications to the expression of a protein that are not encoded by the sequence of its
gene alone. Posttranslational modification can bring about change in parameters such as molecular weight and
the isoelectric point of proteins.
• 2 D-E are extensive, particularly in regard to detecting and quantifying modification in genome expression
during development under different environmental and stress conditions. If the modified group can be
removed by chemical or enzymatic treatment, then 2D gels can be used to identify the position of the modified
proteins.
Applications of 2D-E cont..

49. 3. Protein – Protein interactions
4. Metabolic engineering
Applications of 2D-E
• Proteins within the cells often interact with small molecules, nucleic acids and/or other proteins.
• 2-DE, has been found to be especially useful in the study of protein-protein interactions, because it allows
an improved separation of proteins as well as the detection of specific interacting protein isoforms of a
protein that arise from posttranslational modification.
• It is obvious that all proteins of a living cell are not expressed simultaneously. This makes it possible to
compare the proteome maps under different conditions. The up-regulated and down-regulated proteins
which are stimulated by withdrawal of nutrients, or any other physical and chemical changes, can be
determined by 2-DE. In this way proteins of high significance are found.

50. Applications of 2D-E cont..
5. Characterization of mutant proteins
6. Microbial systematics and
epidemiology
• The characterization of mutant proteins in response to elucidation of regulatory proteins and pathways has been
greatly aided by the use of 2D-gel electrophoresis. The application of this technique to this area of research in
microbial physiology has been extensive and has managed to unravel many regulatory components associated
with bacterial metabolism. For example, this gel-based differential proteomics study enabled to elucidate an
example of global regulation in the model bacterial strain B. subtilis affected by a gene deletion (Seul et al., 2011).
Seul, K. J., Cho, H. S., Ghim, S. Y. (2011). Characterization of a PyrR-deficient Mutant of Bacillus subtilis by a Proteomic Approach. Korean Journal of Microbioliogy and B iotechnology , Vol. 39, No. 1, pp. 9-19.
• The proteome of different strains of bacteria can be analyzed to find the biomarkers related to various diseases.
This technique provides a high resolution & large-scale screening for differentiation of bacterial isolates. For this
reason, a microbial proteomics database system was set up at the Max Planck Institute for Infection Biology
based on 2DE/MS. Many studies have been undertaken to detect biomarkers for various conditions using
differential proteomics, for example the Haemophilus influenzae proteome was analyzed by 2-DE and extended
to three other members of the Haemophilus genus in 1997. The E. coli isolates collected from clinical specimens
were also discriminated by 2-DE..

51. Applications of 2D-E cont..
7. Evaluation of proteins involved in the toxic response and Vaccine development
• The changes in the patterns of in vivo gene expression of pathogens have been revealed using 2-DE
combined with metabolic radiolabelling or immunoblotting.
• The expression pattern of proteins at higher levels in intracellular bacteria is generally similar to
those induced in bacteria in response to stress conditions including extreme acidity, oxygen and
high temperature.
• It is possible to compare uninfected and infected whole protein patterns using 2-DE.
• The identification of immunogenic proteins by using 2- DE, immunoblotting and polyclonal sera will
significantly aid in vaccine development.
• Proteomic/immunoproteomic analyses of H. pylori, Neisseria menangitidis, Streptococcus
pyogenes, Bacillus anthracis and M. tuberculosis have been carried out to find new potential
vaccine candidates using 2-DE.