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Abbasi protein microarray

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  • 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 ◦ 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
  • 3. Types of Arrays Analytical Microarrays Functional Microarrays Reverse Phase Microarrays
  • 4.  Profiles Mixture of Proteins Measure Binding Affinity ◦ Specificity ◦ Protein Expression Levels ◦   Most Common 3 main probe types Antibodies ◦ Affibodies ◦ Aptamers ◦
  • 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.
  • 6. Plates  Full length proteins & protein domains  Functional Samples  Purified & Labeled  Nucleic Acids  Proteins  Lipids  Small Molecules
  • 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
  • 8.  Plates ◦  Cell Lysate Sample ◦ Antibodies of interest  Primary  Attach to spots ◦ Secondary  Attach to primary  Labeled  Detect Altered Proteins Post-translation modification problems ◦ Disease ◦
  • 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.
  • 13.    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      
  • 14. Random Attachment Adsorption Covalent cross linking Uniform Attachment Affinity binding Zhu, H; Snyder, M: Current Opinion in Chemical Biology, 2003, 7, 55-63
  • 15. Red dots indicates active sites on proteins Zhu, H; Snyder, M: Current Opinion in Chemical Biology, 2003, 7, 55-63
  • 16. www.functionalgenomics.org.u k