Protein Microarray Part 1: Introduction, Probes on Chip, sources of probes and detection system
#biotechnology #protein #microarray #chips
Arrays are solid supports upon which a collection of gene-specific probes have been placed at defined locations, either by spotting or direct synthesis.
The term “micro” refers to the small size of the solid support
There are two major types of Arrays
DNA microarray An indirect method for proteomics
Protein Microarray A direct method for proteomics
A Protein microarray (or protein chip) is a high-throughput method used to track the interactions and activities of proteins, to determine their function, and to determine function on a large scale. Its main advantage lies in the fact that large numbers of proteins can be tracked in parallel.
The Probes on Chip
A variety of materials can be immobilized on the protein chip based on the specific requirements. These include:
a. Antibodies
b. Antigens
c. Aptamers (Nucleic Acid-based ligands)
d. Affibodies (small, robust proteins engineered to bind to a large number of target proteins or peptides with high affinity, imitating monoclonal antibodies, and are therefore a member of the family of antibody mimetics)
e. Full-length Proteins or their domains
The Sources of Probes on Chip
1. Cell lysates: By using different chromatographic techniques like affinity chromatography, Ion exchange chromatography etc
2. Recombinant DNA Technology: Utilizing expression systems
3. Synthetic Peptides: The established method for the production of synthetic peptides in the lab is known as Solid-Phase Peptide Synthesis (SPPS).
4. Cell-free translation system: Cell-Free Protein Synthesis (CFPS), also known as in vitro protein synthesis, is the production of protein using biological machinery in a cell-free system, that is, without the use of living cells. Common components of a cell-free reaction include a
Cell extract
An energy source [Phosphoenol pyruvate, acetyl phosphate and creatine phosphate],
Supply of amino acids
Cofactors, such as Mg2+ [for keeping the ribosome intact]
The DNA with the desired gene.
The necessary cell machinery including ribosomes, aminoacyl-tRNA synthetases, translation initiation, and elongation factors, etc
The most famous systems are
a. Nucleic Acid Programmable Protein Array (NAPPA)
b. Protein in situ array (PISA)
Protein Immobilization on a Solid Support
Following protein production, protein immobilization on solid support (e.g. glass slides) is also crucial. The most common supporting materials in use incudes Aldehyde, carboxylic ester, nitrocellulose membrane, polystyrene, Agarose/polyacrylamide gel, hydrogel.
An ideal surface for protein microarray fabrication has to be capable of
a. Immobilizing proteins
b. Preserving three‐dimensional (3‐D) conformation of the protein
c. Should not change the chemical nature of the protein
An aqueous environment is essential at all stages of array manufacture and operation to prevent protein denaturation. Therefore, sample buffers are used to provide an optimum environment to proteins and the humidity of the manufacturing environment is carefully regulated. Micro-wells have the dual advantage of providing an aqueous environment while preventing cross-contamination between samples.
The detection system used in protein microarray is like the ones used in DNA microarray
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