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