1 / 26

Protein microarrays

Protein microarrays. Martyna Franczuk. Adriana Lagraulet , J ournal of the Association for Laboratory Automation (2010). Protein arrays. 25 000 genes and 1 milion proteins! Enable functional protein analysis (in contrast with traditional protein profiling).

caldwell-porter
Download Presentation

Protein microarrays

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Protein microarrays Martyna Franczuk

  2. Adriana Lagraulet, Journal of the Association for Laboratory Automation (2010)

  3. Protein arrays 25 000 genes and 1 milion proteins! • Enable functional protein analysis (in contrast with traditional protein profiling).

  4. Marina Cretich , Francesco Damin, Giovanna Pirri, Marcella Chiari, Biomolecular Engineering (2006)

  5. Three types of protein microarrays: (1) analytical protein microarrays (2) functional microarrays (3) reverse phase microarrays

  6. Analytical protein microarrays • Constructed by arryingantibodies, aptamers,affibodiesornonfoldedantigens on theglassslide. • Based on thesandwich ELISA methodology. • Used to profiling protein mixtures: • binding affinities, • specificities, • protein expression levels. • Most common – antobodymicroarrays • Monitoringdifferential expression profiles and clinical diagnostics

  7. Functional microarrays • Contain full-length functional proteins or protein domains attached to porous polymer matrix. • Used for studying protein interactions with other macromolecules or small molecules and for testing entire proteome biochemical activities.

  8. Reverse phase microarrays • Cell lysate or plasma are arrayed onto a glass or nitrecellulose slide. • Probing with antibodies against the protein of interest. • evaluation of protein expression levels, determination of the presence of altered proteins and dysfunctional protein pathways

  9. Claudius Mueller, Lance A. Liotta, Virginia Espina, Molecular Oncology(2010)

  10. Protein chip preparation • Obtaining proteins from protein libraries • immobilizing proteins onto a treated microscope slide using a contact spotter or a non-contact microarrayer

  11. Protein chip preparation David A. Hall, JasonPtacek, and Michael Snyder, Mechanisms of Ageing and Development (2007)

  12. Protein chip preparation • Random attachment and passiveadsorbtion: glass surface with nitrocellulose, gel pads, or poly-L-lysine • Random attachment: amines, aldehyde- and epoxy-derivatized glass surfaces • Uniform orientation: affinitytagsurfaces • Aqueous environment: epoxy-traetedmicrowells • Direct protein production on chips

  13. Detection of molecule–protein interactions • Label-based • covalent conjugation of N-hydroxysuccinimide ester-linked fluorescent dyes to primary amines of the protein N-terminus or by thiol-reactive dyes to cysteine residues • sandwich ELISA detection method • tyramide signal amplification (TSA)

  14. Detection of molecule–protein interactions Problem: possibility that the label itself may interfere with the probe’s ability to interact with the target protein => • Label-free • surface plasmon resonance (SPR), • carbon nanotubes, carbon nanowires, and microelectromechanical systems cantilevers

  15. Applications • Discovery of drug targets. • Sera screening for the presence of autoantibodies or viral specific antibodies. • Cancer and pathogen diagnostic.

  16. Protein array technology to detect HER2 (erbB-2)-induced‘cytokine signature’ in breast cancer Alejandro Vazquez-martina, Ramon Colomera, Javier A. Menendeza European Journal of Cancer 4 3 ( 2 0 0 7 )

  17. Introduction • HER2 overexpression and breast cancer • Human Cytokine Array III • Media from MCF-7 breast cancer cell lines and plasma from patients • Interleukin-8 nad Growth-related oncogene (GRO) may represent a ptahway involved in tumor progression

  18. (a) Template showing the location of cytokine antibodies spotted onto the RayBioTM Human Cytokine Array III.

  19. Pharmacological inhibition of HER2-drivencellular signalling knock-down IL-8 and GRO expression

  20. Detection of cytokine expression from metastatic breast cancer (MBC) patients sera.

  21. High Diagnostic Accuracy of AntigenMicroarray for Sensitive Detection ofHepatitis C Virus Infection Jung-ah Kwon, Hyeseon Lee, Kap No Lee, Kwangchun Chae, Seram Lee, Dong-ki Lee, and Soyoun Kim Clinical Chemistry 54 (2008)

  22. Introduction • Confirmatory test for blood serodiagnosis • 96-well antigen microarray on sol-gel derived material • Patients sera samples previously tested for HCV by ELISA • Assay time and conditions compatible with conventional ELISA diagnostic assay

  23. Literature • Adriana Lagraulet, Current Clinical and Pharmaceutical Applications of Microarrays: From Disease Biomarkers Discovery to Automated Diagnostics[in:] Journal of the Association for Laboratory Automation15 (2010), 405-413. • Marina Cretich , Francesco Damin, Giovanna Pirri, Marcella Chiari, Protein and peptide arrays: Recent trends and new directions[in:] Biomolecular Engineering 23 (2006), 77–88. • David A. Hall, JasonPtacek, and Michael Snyder, Protein microarray technology [in:] Mechanisms of Ageing and Development 128 (2007), 161–167. • Claudius Mueller, Lance A. Liotta, Virginia Espina, Reverse phase protein microarrays advance to use in clinical trials[in:] Molecular Oncology4 (2010), 461-481. • Presentedoriginalarticles

More Related