1. Protein arrays as tool for studies at the
host pathogen interface
Professor: Dr Habibi
By: Abbas Abbasy Rooholahi
NBT_FALL_1392
2. Similar to DNA microarrays
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Plate, Probe, Attachment
Advantage
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Poor correlation between mRNA and protein
expression
Study protein interactions
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Protein-Protein
Protein-Ligand
Protein-DNA
Monitor Disease States
Clinical Diagnostics
4.
Profiles Mixture of
Proteins
Measure Binding
Affinity
◦ Specificity
◦ Protein Expression
Levels
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Most Common
3 main probe types
Antibodies
◦ Affibodies
◦ Aptamers
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5. 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.
7. 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
9. 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 binding
between a protein printed onto the slide and an
antibody added is revealed by the addition of a
secondary antibody conjugated with a fluorochrome.
10. Key processes and infection strategies during host- patogen intraction
11. A scheme of the main components and interactions in
pathogen-based protein microarrays.
12. Different capture molecules for protein microarray assays.
Recently, nucleic acids, receptors, enzymes, and proteins
have been used as capture molecules.
Capture molecules used are most commonly antibodies. In
antigen-antibody interactions the antigen or a specific
antibody is immobilized and detected on the basis of the
labeled analyte (antigen or antibody) or by labeled secondary
antibodies.
Antibodies have several problems including the fact that
there are not antibodies for most proteins and also problems
with specificity in some commercial antibody preparations.
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14.
15.
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
