Team Members

Saif Ullah (654)

Ahmed Shah (602)
Low Asnan Amin (608)
Throughput Muhammad Talha (634)
Muhammad Younas (638)
Method Nadeem Anwar (656)
Ameer Muhammad (604)
Proteomics
Introduction
 Proteomics:
 Study of Proteome, Investing how Different Protein Interact with each other & the
Roles they play with in the Organism.
 Proteome:
 Set of Proteins, Expressed in a Cell at a Specific Time.
 The word Proteome was coined by a Australian Ph.D. Student Marc Wilkin in 1994, in
a Symposium Held in Siena (Italy).
 History:
 The First Experiment of Proteomics is Done with the Development of 2D Protein
Electrophoresis in 1975.
 However, Truly High Throughput Identification of Multiple Proteins Per Sample with
the Development of Mass Spectrometry (MS) Tech over 20 Years.
Proteomics
Introduction
 Why We Study Proteomics
 Unlike the Genome, the composition of a Proteome is in Constant State of Flux Over
Time & throughout the Organism.
 Proteome Keeps Changing:
 Temporary Guided: Expression of a Proteome at a Given Point of Time.
 Separately Guided: Expression of a Proteome of a Particular Cell Type/Tissue within the
Organism doesn’t Match with Other Organisms.
 Proteomics Can Answer Following Questions:
 Protein Identification, Protein Quantification, Protein Localization, Post-Translational
Modifications, Protein-Protein Interaction etc.
Proteomics
Methods Involved in Proteomics:
 Low Throughput Method
 Such Techniques are called as Low Throughput Methods that Generates Less Amount
of Data.
 High Throughput Method:
 Such Techniques that Generates Large Amount of Data
ELISA
Principle of ELISA
 Antibody is Immobilized on Micro-Plate Wells.
 Competition Between in Sample & Labeled Enzyme for Antibody Binding Sites.
 The Unbound Material is Washed Out.
 Chromogenic Substrate added to Develop Color.
 Resulting Color is Read in a Spectrophotometer.
ELISA
Introduction
 ELISA, or Enzyme-Linked Immunosorbent Assay, is an Immunoassay Technique
involving the Reaction of Antigen & Antibody in Vitro.
 ELISA is a Sensitive & Specific Assay for the Detection & Quantitation of
Antigens or Antibodies.
 ELISA Tests are usually performed in Microwell Plates.
 The ELISA test, or the Enzyme Immunoassay (EIA), was the First Screening Test
commonly employed for HIV. It has High Sensitivity.
ELISA
Direct ELISA
 Apply a Sample of Known Antigen to a Surface.
 Enzyme Linked Primary Antibody is Applied to the Plate.
 Washed, After this Wash, Only the Antibody-Antigen Complexes Remain
Attached.
 Apply a Substrate which is Converted by the Enzyme to Elicit a Chromogenic
Signal.
ELISA
Sandwich ELISA
 Plate is Coated with suitable Antibody.
 Blocking Buffer is added.
 Sample is Added to Plate, so Antigen is Bounded by Capture Antibody.
 A suitable Biotin Labeled Detection Antibody is added to Plate.
 Enzyme HRPO is added & Binds the Biotin Labeled Detection Antibody.
 TMB Substrate is added and converted by HRPO to Colored Product.
ELISA
Sandwich ELISA Test
ELISA
Indirect ELISA
 In Indirect ELISA, A Primary Unlabeled Ab binds with Immobilized Ag & is in turn Bound to
a Secondary Enzyme-Linked Ab, which produces Color change on addition of a suitable
substrate.
 It is used to detect the Presence of an Ab.
 It involves the following Steps:
 Coating Surface with Ag
 Washing off Excess Ag
 Addition of Primary Ab
 Binding of Ag-Primary Ab
 Washing off Excess Ab
 Addition of Secondary Ab
 Washing off Excess Ab
 Addition of Substrate
 Color Change & Quantification by Spectrometry
ELISA
Advantages of ELISA
 Reagents are Relatively Cheap & Have a Long Shelf Life
 ELISA is Highly Specific & Sensitive
 No Radiation Hazards occur during Labelling or Disposal of Waste
 Easy to Perform & Quick Procedures
 Equipment can be Inexpensive & Widely Available
 ELISA can be used to a Variety of Infections
ELISA
Disadvantages of of ELISA
 Measurement of Enzyme Activity can be more Complex than Measurement of
Activity of Some Type of Radioisotopes.
 Enzyme Activity may be Affected by Plasma Constituents.
 Kits are Commercially Available, but Not Cheap.
 Very Specific to a Particular Antigen, won’t Recognize any other Antigen.
 False Positives/Negatives Possible, especially with Mutated/Altered Antigens.
2-D Gel Electrophoresis

 2-D Electrophoresis is a Powerful & Widely used method for the analysis of
Complex Protein Mixtures extracted from Cells, Tissues, or other Biological
Samples.
 It’s the method available which is capable of Separating thousands of Proteins.
 This Technique Separates Proteins in Two Steps
1. First-Dimension is Isoelectric Focusing (IEF)
 Separates Proteins according to their Isoelectric Points (pI).

2. Second Dimension is SDS-Polyacrylamide Gel Electrophoresis (SDS-Page)

 It Separates Proteins according to their Molecular Weight (MW).
2-D Gel Electrophoresis
Principle (Isoelectric Focusing):
 IN 2D Gel Electrophoresis Proteins are Separated as per Isoelectric Point and
Protein Mass.
 Separation of the Proteins by Isoelectric Point is called Isoelectric Focusing (IEF).
 When a Gradient of pH is applied to a Gel & an Electric Potential is Applied across
the Gel, Making One End more Positive than Other.
 At all pH Values other than their Isoelectric Point, Proteins will be charged. If they
are Positively Charged, they will be Pulled towards the Negative End of the Gel.
 In a pH Gradient & under the influence of an Electric Field, a Protein will move to
the Position in the Gradient where its Net Charge is Zero.
 If a Protein diffuse away from its pI, it immediately Gains Charge & Migrates Back.
This is the Focusing Effect which allows the Proteins to be Separated on the basis of
Very Small Charge Differences.
2-D Gel Electrophoresis
Principle (2D Electrophoresis):
 In Separating the Proteins by Mass, the Gel treated with Sodium Dodecyl Sulfate (SDS)
along with other Reagents (SDS-PAGE).
 This Denatures the Proteins (Unfolding them into Long, Straight Molecules) & binds a
Number of SDS Molecules roughly Proportional to Protein’s Length. Because a
Protein’s Length (When Unfolded) is Roughly Proportional to its Mass.
 In Addition, Proteins will not Migrate when they have No Charge (A Result of the
Isoelectric Focusing Step). Therefore, the Coating of the Proteins in SDS (Negatively
Charged) allows Migration of the Proteins in the Second Dimension.
 In Second Dimension, an Electric Potential is again Applied, but at a 90 Degree Angle
from the First Field. The Proteins will be Attracted to more Positive Side of the Gel
(Because SDS is Negatively Charged).
 The Gel therefore acts like a Molecular Sieve, when the Current is applied, Separating
the Proteins based on their Molecular Weight with Larger Proteins being Retained
Higher in the Gel & Smaller Proteins being able to Pass through the Sieve & Reach
2-D Gel
Electrophoresis Isoelectric Focusing 2D Electrophoresis
Separation Based on Separation Based on
Differences Isoelectric Point Molecular Weight of
Protein
Requires Very High Requires About Voltage
Voltage (10000V) of (200V)
Requires A Long Period Requires A Shorter
of Time (10h) Period of Time (2h)
2-D Gel Electrophoresis
Sample (Precautions):
 Must Break All Non-Covalent Protein-Protein, Protein-DNA, Protein-Lipid
Interactions, Disrupt S-S Bonds.
 Must Prevent Proteolysis, Accidental Phosphorylation, Oxidation, Cleavage,
Deamidation.
 Must Remove Substances that Might Interfere with Separation Process such as
Salts, Polar Detergents, Lipids, Polysaccharides, Nucleic Acids.
 Must Try to keep Proteins Soluble during both Phases of Electrophoresis.
2-D Gel Electrophoresis
Detection / Visualization
2-D Gel Electrophoresis
Applications
 Protein Identification by Mass Profile Fingerprinting
 Due to the High Resolution of 2D Gels, very Simple & Cheap MS Process can be used
to Identify a Protein from a 2D Gel Electrophoresis.
 2D Gel Based Proteomics Analysis Toxicology
 2D Gel Electrophoresis is used to Find an association between Decreased Calcium
Binding Proteins (Calbindin-D 28kDa), Urinary Calcium Wasting & Intratubular
Corticomedullary Calcifications in Rat Kidney.
 2D Gel Based Proteomics in Bacterial Proteomics
 2D Gel Based Proteomics is also Widely Used in Bacterial Proteomics, when the
Complexity of the Sample is Low Enough.
 In Immunoproteomics
 2D Gel Electrophoresis also used in Immunoproteomics, where it is the Immune
Response of Patients that is Probed at a Proteomic Level.
2-D Gel Electrophoresis
Applications
 2D Gel in Post Translation Modification
 2D Gels are also very Appropriate when Post Translation Modifications are Studied.
 Many Post Translation Modifications do Alter the Isoelectric Point or the Molecular
Weight of the Proteins and thus induce Position shifts in 2D Gel.
 Cell Response to Oxidative Stress
 The Proteomics analysis reported shows that a Major Cellular Response to Oxidative
Stress iss the Modification of Several Peroxiredoxins.
2-D Gel Electrophoresis
Disadvantages of 2D Gel Electrophoresis
 This Technique include a Large Amount of Sample Handling.
 Limited Reproducibility & a Smaller Dynamic Range than some other
Separation Methods
 Difficulty in Separation of Hydrophobic Proteins.
 It is also not Automated for High Throughput Analysis
 Certain Proteins are Difficult for 2D-PAGE to Separate, Including those that are
in Low Abundance, Acidic, Basic, Very Large or Very Small.
HPLC (High Performance Liquid Chromatography)
Introduction
 High-Performance Liquid Chromatography or commonly known as HPLC, is an
Analytical Technique used to Separate Identify or Quantify each Component in a
Mixture.
 The Mixture is Separated using the basic Principle of Column Chromatography
& then Identified & Quantified by Spectroscopy.
 HPLC is basically a Highly Improved form of Column Liquid Chromatography.
 Instead of Solvent being Allowed to Drip through a Column under Gravity, it’s
Forced through Under High Pressures of Up to 400 Atmospheres.
HPLC (High Performance Liquid Chromatography)
Principle of HPLC
 The Purification takes place in a Separation Column between a Stationary & a
Mobile Phase.
 The Stationary Phase is a Granular Material with Very Small Porous Particles in
a Separation Column.
 The Mobile Phase, on the other hand, is a Solvent or Solvent Mixture, which is
Forced at High Pressure through the Separation Column.
 Via a Valve with a Connected Sample Loop, i.e. A Small Tube or A Capillary
Made of Stainless Steel, the Sample is Injection into the Mobile Phase Flow from
the Pump to the Separation Column Using a Syringe.
 Individual Components of the Sample Migrate through the Column at Different
Rates because they are Retained to a Varying Degree by Interactions with the
Stationary Phase.
 After Leaving the Column, the Individual Substances are Detected by a Suitable
HPLC (High Performance Liquid Chromatography)
Principle of HPLC
 At the End of this Operation/Run, a Chromatogram in the HPLC Software on
the Computer is Obtained.
 The Chromatogram Allows the Identification & Quantification of the Different
Substances.
HPLC (High Performance Liquid Chromatography)
Applications of HPLC

 Analysis of Drugs
 Analysis of Synthetic Polymers
 Analysis of Pollutants in Environmental Analytics
 Isolation of Valuable Products
 Water Purification
 Product Purity & Quality Control of Industrial Products & Fine Chemicals
 Separation & Purification of Biopolymers, such as Enzymes or Nucleic Acids.
HPLC (High Performance Liquid Chromatography)
Advantages & Disadvantages of HPLC
 Advantages:
 Speed
 Efficiency
 Accuracy
 Versatile & Extremely Precise, when it comes to Identifying & Quantifying
Chemical Components.
 Disadvantages:
 Cost: HPLC can be Costly, Requiring Large Quantities of Expensive Organics.
 Complexity
 HPLC does have Low Sensitivity for Certain Compounds & Some Cannot be Detected
as they are Irreversible Adsorbed.
TEAM:
THE BOYS 