Protein arrays as tool for studies at the host pathogen interface

Professor: Dr Habibi

By: Abbas Abbasy Rooholahi

NBT_FALL_1392

Similar to DNA microarrays◦ Plate, Probe, Attachment

Advantage◦ Poor correlation between mRNA and protein

expression

Study protein interactions◦ Protein-Protein

◦ Protein-Ligand

◦ Protein-DNA

Monitor Disease States

Clinical Diagnostics

Analytical Microarrays

Functional Microarrays

Reverse Phase Microarrays

Types of Arrays

Profiles Mixture of Proteins◦ Measure Binding

Affinity

◦ Specificity

◦ Protein Expression Levels

Most Common

3 main probe types◦ Antibodies

◦ Affibodies

◦ Aptamers

The molecule of interest, an antibody or an antigen, is printed onto the

slide. After the hybridization with the sample, the interactions can be

detected through different strategies .

Antigen arrays are employed as platforms to monitor humoral immunity

Antibody microarray based technology is a powerful emerging tool in

proteomics, target discovery and differential analysis, and has been

mainly used for the identification of protein expression signatures

related to infection and for serum protein profiling studies.

Plates

Full length proteins & protein domains Functional

Samples

Purified & Labeled Nucleic Acids

Proteins

Lipids

Small Molecules

Functional microarrays are based on printing full length

functional proteins or protein domains to study the

biochemical characteristics and functions of native proteins.

Peptides or domains which are highly purified through cell

based methods or by cell-free expression on the microarray

They can evaluate, validate and monitor protein in a cost

effective manner and address the issue of a high quality

protein supply to be used in the array being accurately

employed to identify and study the diverse protein interactions

protein– protein, protein–DNA, protein–RNA, protein–

phospholipids and protein–small molecules

Plates◦ Cell Lysate

Sample◦ Antibodies of interest Primary Attach to spots

◦ Secondary Attach to primary Labeled

Detect Altered Proteins◦ Post-translation

modification problems◦ Disease

In RPA, cellular or tissue lysates are immobilized on the

slides.

An ELISA approximation is used to reveal the ligand–

protein interaction and a fluorochrome is employed in order

to detect such interaction.

Various antibodies are faced to the slide; the bindingbetween a protein printed onto the slide and anantibody added is revealed by the addition of asecondary antibody conjugated with a fluorochrome.

Key processes and infection strategies during host- patogen intraction

A scheme of the main components and interactions in

pathogen-based protein microarrays.

Different capture molecules for protein microarray assays.

Recently, nucleic acids, receptors, enzymes, and proteinshave been used as capture molecules.

Capture molecules used are most commonly antibodies. Inantigen-antibody interactions the antigen or a specificantibody is immobilized and detected on the basis of thelabeled analyte (antigen or antibody) or by labeled secondaryantibodies.

Antibodies have several problems including the fact thatthere are not antibodies for most proteins and also problemswith specificity in some commercial antibody preparations.

Acrylamide and agarose – capture antibodies and proteins

Plain Glass Slides

3D Gel Pad chip Water environment

Reduce evaporation

Minimizes cross-contamination

Change of buffer

Recovery of trapped molecules

Nanowells - polydimethylsiloxane surface (PDMS) Multiplexing

Easy removal of captured molecules

Specialized equipments required

Random Attachment

Adsorption

Covalent cross linking

Uniform Attachment

Affinity binding

Zhu, H; Snyder, M: Current Opinion in Chemical Biology, 2003, 7, 55-63

Red dots indicates active sites on proteins

Zhu, H; Snyder, M: Current Opinion in Chemical Biology, 2003, 7, 55-63

www.functionalgenomics.org.uk