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High throughput Protein Measurement Techniques

Explore high-throughput protein measurement techniques such as Proteinchip, Protein Array, and Microarray for biological research, biomarkers, drug discovery, and system response profiling. Overcome bottlenecks in capture agents, purification, labeling, and concentration. Begin a journey of advanced protein analysis.

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High throughput Protein Measurement Techniques

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  1. High throughput Protein Measurement Techniques Harin Kanani

  2. Proteins: • Biological Macromolecules • Functional Protein - Enzymes • Structural Protein • Antibody for Cell defense • Signalling Proteins

  3. Motivation for Protein measurement: • Basic biological research • Biomarkers : Molecular Classification & Diagnosis of disease • Drug Discovery • For profiling response of biological systems to toxins and pharmaceuticals

  4. Motivation High throughput Measurement: ::Genomic Revolution:: • Rapid (?) measurement of a large number of samples • non-hypothesis approach of research • Final Aim is - “Simultaneous, Complete, quantitative analysis of all Gene Expressions/ Proteins”

  5. High throughput Measurement: Proteomics Genomics • Proteinchip • Protein Array Metabolomics • Genechip • DNA Microarray • GC-LC / MS • NMR

  6. High throughput Measurement: Proteins Proteomics Genomics • Proteinchip • Protein Array Metabolomics • Genechip • DNA Microarray • GC-LC / MS • NMR

  7. ProteinchipTM Protein Capture Chips for Mass Spectrometry Using SELDI – Surface Enhanced Laser Disorption Ionization Or MALDI – Matrix Assisted Laser Disorption Ionization

  8. Protein Capture-chips:

  9. ProteinchipTM : Procedure Application of Crude Sample: Step:1

  10. ProteinchipTM : Procedure Wash chip to remove non-specifically binded protein/background: Step: 2

  11. Protein Capture-chips – MS: Procedure Add Energy Absorbing Molecules (EAS) or “Matrix”: Step: 3 This step loosens the protein binding

  12. Protein Capture-chips – MS: Procedure Use Laser for desorption and ionization of protein : Step: 4 TOF (Time of Flight Mass Spectrometer): Measure Molecular weight – Get Spectrum

  13. Protein Capture-chips – MS: Results Generating m/z vs Intensity Plots Step: 5

  14. Protein Capture-chips – MS: Results

  15. ProteinchipTM CIPHERGEN Proteinchip Biology System

  16. Protein Microarray High-throughput Quantification (Profiling) using specific antibody – antigen Interaction & relative intensity of floroscent signal

  17. Protein Microarray Technology:Slide Chemistry SlidePreperation Protein Imobilization Quenching Additional sites bovine serum albumin (BSA)

  18. Protein Microarray Technology:Protein Detection

  19. Protein Microarray Technology:Intensity vs. Protein Concentration

  20. Protein Microarray Expression Profiling Sample Control

  21. Protein Microarray Technology: Microarrayer Scanning Image Analysis Software Measure intensity with Cy-3 and Cy-5 Filter

  22. Protein Microarray Technology: Relative Protein Abundance In a sample Data Normalization Statistical Techniques To compare Relative Abundance in different samples Experimental Conclusion/ Discoveries

  23. Microarrayer : AFFIMATRICS GMS-417 Arrayer

  24. Proteine Microarray:

  25. Proteine Microarray:

  26. Commercial Protein Measurement Technique:

  27. Commercial Protein Measurement Technique: Many Techniques being explored… but very few Commercial products in the market Inspite of high demand Because……

  28. Protein Measurement: Bottlenecks.. • Capture agents: shortage of sufficiently specific antibodies or their ligands • Problem due to different forms of the post translational protein • Protein deactivation on micro array surfaces

  29. Protein Measurement: Bottlenecks.. • High throughput purification techniques which do not affect functional form of proteins • lack of uniform labelling due to different protein properties ..(upto 10 fold difference) • Varying protein concentration (~10^14) ..different concentration requires different conditions

  30. High throughput Protein Measurement Beginning of a long journey ahead….

  31. Thank You

  32. References: • Mitchell P, “ A perspective on protein microarray”, Nature Biotechnology, 20, 225 • Haab B., et. al., “ Protein microarray for highly parallel detection and quantitation of specific proteins and antibodies in complex solutions”, Genome Biology 2001, 2(2):research/004.1 • Proteinchip Publications, literature and posters on: • www.ciphergen.com • MacBeath et al., “Printing Proteins as Microarrays for High-Throughput Function Determination”, SCIENCE, 289, 1760 • Zhu et. al., “ Global analysis of protein activities using proteome chips”, SCIENCE, 293, 2101

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