Protein Chips for proteomic Study
-
Upload
antonio-sousa -
Category
Technology
-
view
1.813 -
download
0
description
Transcript of Protein Chips for proteomic Study
PROTEIN CHIPSFor proteomic study
António Sousa64427 MBioNano
INDEX1. Introduction2. Traditional Characterization Methods3. History of Protein Chips4. Types of Protein Chips 4.1. Labeling 4.2. Glass slide-based Systems 4.3. Microbead arrays 4.4. Biosensor Chips5. Current Limitations6. Biomedical applications of protein Chips
Genomics has provided insight into genes which play roles in specific biological processes, diseases, and drug treatments.
Proteomics is at least equally important both in biomedical research and clinical diagnostics Since proteins play a key role in phenotype development.
1. INTRODUCTION
2. TRADITIONAL PROTEIN CHARACTERIZATION METHODS
2-DE
• Comparative studies of different samples, such as normal versus diseased, or treated versus untreated, to determine expressional differences.
ELISA, IH
• Due to its sensitivity and reliability. capable of localizing proteins of interest within a cell or tissue utilizing specific visualization techniques, such as fluorescently labeled antibodies
MS
• Proteins can be affixed to various special affinity surfaces or embedded into an appropriate matrix for concomitant soft ionization.
3. HISTORY OF PROTEIN CHIPS
Western Blotting technology
cDNA
Microarray
technology
Protein Microchips
Originally as large as regular 96-well plates, in such a way that different antibodies were bound in the individual wells of the plates.
Initially, detection was mostly done by radioisotopebased labeling, as it was easily applicable for the analysis of phosphorylated proteins. Detection was by X-ray film exposure
Differential fluorescent dye labeling, brought a real breakthrough to protein labeling and analysis, especially with the advent of cyanine dyes such as the amine-reactive Cy3 and Cy5
4. TYPES OF PROTEIN MICROCHIPS
Protein microchips are high-throughput tools to monitor and determine protein expression levels and investigate interactions of proteins with biologically important molecules
Sample
Substrate with proteins
4.1. LABELING
Direct
Involves binding a fluorescent tag to all analytes.
Indirect
Which uses an antibody specific for the antigen
and a secondary antibody
coupled to a detectable
marker.
RCA
Which is based on specific
immunoreaction, followed by in
synthetic circular DNA replication
4.2. GLASS SLIDE-BASED SYSTEMS
Microfluidic channel system of the Agilent lab-on-a-chip for protein analysis. ©Agilent Technologies.
Protein–protein interactions, most frequently using antibodies that are attached to solid glass surfaces
Labeling systems for planar protein chips is the so-called dye-swap technique, also known as the reverse labeling or the flip flop labeling method.
4.3. MICROBEAD ARRAYS
Illustration of a high-density microbead
FlowCytometr
y
μBead μBead μBead
AttachedAntibody
Marked protein of interest
Laser Excitation
Microbead-based protein arrays are based on the interaction between surface (polystyrene microspheres) attached capture molecules and proteins of cell lysates. The surface bound complexes on the microbeads are interrogated by flow cytometry.
4.4. BIOSENSOR CHIPS
SPR (Surface plasmon resonance) is one of the most sophisticated methods to detect and quantify biomolecular interactions in real time.
In the case of a biomolecular interaction, the refractive index is altered.
Based on binding pattern changes on a specific sensor surface.
5. CURRENT LIMITATIONS
Sensitivity-related issues
Protein Stability
False positives and background issues
6. BIOMEDICAL APPLICATIONS OF PROTEIN CHIPS
Diagnostics
Drug screening and testing
Disease monitoring
Proteomics and drug discovery
Identification of protein function
Medical Research
ENDAntónio Sousa
64427 MBioNano