Protein microarray
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This document discusses protein microarrays, which allow high-throughput analysis of thousands of protein interactions. It describes the basic principles and experimental process of protein microarrays, including sample preparation, printing, incubation, washing, and data analysis. Protein microarrays have applications in detecting protein binding properties, profiling antibody specificity, studying post-translational modifications, and identifying biomarkers for clinical research applications like cancer. While powerful for proteomics research, protein microarrays also have some limitations like high costs.
Protein microarray
- 1. PROTEIN MICROARRAY Ghalia Nawal Roll#33 MS I Biotechnology 2015-2017
- 2. What is Microarray? Miniaturization of assays History Protein Microarray 30,ooo-250,000
- 3. Principle: Protein microarrays constitute a powerful tool for high throughput and multiplexed protein analysis including protein detection, investigation of protein interactions with various types of molecules and determination of protein functions. This technology is highly sensitive, highly parallel (multiplexed), generates large amount of data in single experiment with comparatively low sample consumption so is highly economical and facilitate the parallel screening of thousands of interactions, encompassing protein anti-body, enzyme substrate, protein ligand or protein drug and protein –protein interactions.
- 5. Sample Preparation Obtain 0.2-1.0 μg/μl of protein Sample Transfer 4 μl of each into a well microplate First Priniting buffer (final protein spotting conc. of 0.1-0.5 μg/μl) Stabilize at 4 Degrees. Mixing(10 times pipetting)addition of 2x Blocking buffer Print slides ( at 20°C and a relative humidity of 45-60%) Dry at room temperature for 15-30 min. Transfer to wash station ,Incubate 30-60 mins, with vigorous agitation Wash the printed microarrays (remove unbound protein+comp.of Printing Buffer) The processed microarrays containing coupled target proteins is now ready reacting with florescent Dye.
- 6. Biochemical Reaction • Add 10 μl of labeled detection reagent (cysteine residues) to each 10 μl protein sample • Incubate (times range from 30 min to 3 hrs) • Mix the samples thoroughly by pipetting up and down 10 times prior to application on the array.
- 7. Data Analysis and Results • Scan the microarray to produce the microarray image. The dried slide can be read with any standard fluorescence-based microarray scanner . It is physically impossible for an individual to accurately monitor the interaction occurring on several hundreds or thousands of individual spots. In addition, while conducting statistical analysis and group comparison, several degrees of data sets are involved. Although advanced data analysis Software like QArray software is available but it is still difficult to accurately comprehend the biological significance of the data obtained. In Addition, regression analysis software, particularly for kinetics study are used.
- 8. illustrations of step by step Arraying instructions of QArray software.
- 9. Protein Microarray Fabrication Protein Production Surface Chemistry Detection
- 10. Categories Two major classes of protein microarrays are defined to describe their applications: • Analytical protein microarrays • Functional protein microarrays In addition, tissue or cell lysates can also be fractionated and spotted on a slide to form • Reverse-phase protein microarray.
- 11. Representative model APM is the antibody array Cancer cell development research Slides are prepared by spotting highly purified antigens onto special nitrocellulose- coated slides, the arrays are incubated, treated with a secondary fluorescence labeled antibody (Cy3, Cy5) for visualizing the antibody-antigen interactions
- 12. constructed using individually purified proteins study of various biochemical properties of proteins like binding activities, including :protein-protein, protein-DNA, protein-lipid, protein-drug and protein-peptide interactions, and enzyme-substrate relationships via various types of biochemical reactions .
- 13. Many different probes can be tested to specifically identify certain proteins in lysate samples monitor histological changes in prostate cancer patients
- 14. Table 2: Commercially available Protein Microarray
- 15. APPLICATIONS Detection of Protein Binding Properties: Protein-protein interaction Protein-Peptide Interaction Protein-DNA Interaction Small Molecule Interaction Protein-Glycan Interaction Profiling antibody specificity Post-translational modifications: Protein phosphorylation Protein Ubiquitylation Protein Acetylation S-Nitrosylation Clinical Research Host Microbe Interaction Biomarker Identification Current Research in Cancer Future Prospects!!!!!
- 16. Pros Cons • more sensitive, reliable and efficient method for small molecule detection than traditional ELISA • Protein microarrays have the potential to replace singleplex analysis systems • Greater potential in proteomics than any other method . • High Cost $500-1000 per antibody $10 per oligo
- 17. ANY QUESTION
Editor's Notes
- #12: proteins are detected after antibody capture using direct protein labeling