Protein Arrays

Overview
 What Are Protein Arrays?
 General Scheme
 Types of Arrays
◦ Analytical
◦ Functional
◦ Reverse Phase
What Are Protein Arrays?
 Similar to DNA microarrays
◦ Plate, Probe, Attachment
 Advantage
◦ Poor correlation between RNA and protein expression
 Study protein interactions
◦ Protein-Protein
◦ Protein-Ligand
◦ Protein-DNA
 Monitor Disease States
 Clinical Diagnostics
Protein Microarrays

A high density array containing 100s

to many thousands of proteins
positioned in an addressable
format
 Protein Microarray

1. High throughput
analysis of hundreds of
thousands of proteins.
2. Proteins are
immobilized on glass.
3. Various probes
(protein, lipids, DNA,
peptides, etc) are used.
General Scheme
 Types of Arrays
 Analytical
Microarrays
 Functional
Microarrays
 Reverse Phase
Microarrays
 Analytical Array
 Probes (antibody) on surface recognize
target proteins.
 Identification of expressed proteins from
samples.
 Typical quantification method for large # of
expressed proteins.
Analytical Microarrays
 Profiles Mixture of
Proteins
◦ Measure Binding Affinity
◦ Specificity
◦ Protein Expression
Levels
 Most Common
 3 main probe types
◦ Antibodies
◦ Affibodies
◦ Aptamers
 Plate Set Up
 Choose plate surface
◦ Glass, Silicon
 Attachment Method
◦ Random Attachment
 Covalent attachment by amines
 Aldehyde
 Epoxy
 Adsorption
 Nitrocellulose
 Poly-L-Lysine
 Acrylamide Gel Pads
◦ Uniform Attachment
 Affinity Tag
 Nickel Coat & His tag
 Streptavidin & Biotin
 Spots vs. Wells
 Sample incubated on plate with
probes
Analytical Microarray Plates
 Antibodies
◦ 150 kDa
◦ Standard
 Affibodies
◦ non-immunoglobulin-based
affinity reagents
◦ Based on Staphylococcus
aureus protein A
 Alpha-helices
 No Disulfide
 6 kDa
◦ Randomization of 13 AA in
binding domain
Plates continued
 Aptamers
◦ Nucleic Acids
 DNA, RNA, etc.
◦ Peptides
 Variable loop (10-20 AA)
 Protein Scaffold
◦ Bind Protein
 Van der Waals Forces, H
bonding, Electrostatic
Interaction
◦ Highly Specific
◦ Engineered completely
in test tube
 In vitro selection
 Sample Preparation
 Sample extracted
from cells or tissues
 Bio-Rad assay
 Labeled
◦ Fluorescent Dye
 Cy3/Cy5 via Lysines
◦ Photochemical
◦ Radioisotope
◦ May interfere
Detection & Quantification
 Scanner
◦ Detects dye
◦ Adjusts for
background
 Reference spots
◦ Labeled known
concentrations
 Computational
Analysis
 Functional Microarrays

 Plates
◦ Full length proteins &
protein domains
 Functional
 Samples
◦ Purified & Labeled
 Nucleic Acids
 Proteins
 Lipids
 Small Molecules
 Functional Array

 Probes (proteins) on surface react with

target molecules.
 Reaction products are detected.
 Main goal of proteomics.
Interaction Array

 Probes (proteins, peptides, lipids) on

surface interact with target proteins.
 Identification of protein interactions.
 High throughput discovery of interactions.
Functional Array Example
 Protein-Small
Molecule Interaction
◦ Plate has whole
proteome
◦ Monitor Specificity
◦ Off-target effects
 Reverse Phase Microarrays
 Plates
◦ Cell Lysate
 Sample
◦ Antibodies of interest
 Primary
 Attach to spots
◦ Secondary
 Attach to primary
 Labeled
 Detect Altered
Proteins
◦ Post-translational
modification problems
◦ Disease
 Technical Challenges in Protein
Arrays
1. Poor control of immobilized protein activity.
2. Low yield immobilization.
3. High non-specific adsorption.
4. Fast denaturation of Protein.
5. Limited number of labels – low mutiplexing
General Issues for Standardization

1) Protein content & array platforms vary widely

(Expression systems; slides, etc)
2) Protein quality may vary from prep to prep.
3) Negative results harder to interpret than
DNA arrays
4) Ideally, measurements should be
quantitative
- Ab arrays - Each Ab must be standardized
5) Field is still maturing