Protein microarray - AbsoluteAstronomy.com

A protein microarray, sometimes referred to as a protein binding microarray,
provides a multiplex

Multiplex (assay)

A multiplex assay is a type of laboratory procedure that simultaneously measures multiple analytes in a single assay. It is distinguished from procedures that measure one or a few analytes at a time...

approach to identify protein–protein interactions, to identify the substrates of protein kinase

Kinase

In chemistry and biochemistry, a kinase is a type of enzyme that transfers phosphate groups from high-energy donor molecules, such as ATP, to specific substrates, a process referred to as phosphorylation. Kinases are part of the larger family of phosphotransferases...

s, to identify transcription factor protein-activation, or to identify the targets of biologically active small molecules. The array is a piece of glass on which different molecules of protein

Protein

Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...

or specific DNA binding sequences (as capture probes for the proteins) have been affixed at separate locations in an ordered manner thus forming a microscopic array. The most common protein microarray is the antibody microarray

Antibody microarray

An antibody microarray is a specific form of protein microarrays, a collection of capture antibodies are spotted and fixed on a solid surface, such as glass, plastic and silicon chip for the purpose of detecting antigens...

, where antibodies are spotted onto the protein chip and are used as capture molecules to detect proteins from cell lysate solutions.

Related microarray technologies also include DNA microarray

DNA microarray

A DNA microarray is a collection of microscopic DNA spots attached to a solid surface. Scientists use DNA microarrays to measure the expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome...

s, cellular microarray

Cellular microarray

A cellular microarray, is a laboratory tool that allows for the multiplex interrogation of living cells on the surface of a solid support. The support, sometimes called a "chip", is spotted with varying materials, such as antibodies, proteins, or lipids, which can interact with the cells, leading...

s, antibody microarray

Antibody microarray

s, tissue microarray

Tissue microarray

Tissue microarrays consist of paraffin blocks in which up to 1000 separate tissue cores are assembled in array fashion to allow multiplex histological analysis.-History:...

s and chemical compound microarray

Chemical Compound Microarray

A chemical compound microarray is a collection of organic chemical compounds spotted on a solid surface, such as glass and plastic. This microarray format is very similar to DNA microarray, protein microarray and antibody microarray...

Applications

Protein microarrays (also biochip, proteinchip) are measurement devices used in biomedical applications to determine the presence and/or amount (referred to as relative quantitation) of proteins in biological samples, e.g. blood

Blood

Blood is a specialized bodily fluid in animals that delivers necessary substances such as nutrients and oxygen to the cells and transports metabolic waste products away from those same cells....

. They have the potential to be an important tool for proteomics

Proteomics

Proteomics is the large-scale study of proteins, particularly their structures and functions. Proteins are vital parts of living organisms, as they are the main components of the physiological metabolic pathways of cells. The term "proteomics" was first coined in 1997 to make an analogy with...

research. Usually a multitude of different capture agents, most frequently monoclonal antibodies

Monoclonal antibodies

Monoclonal antibodies are monospecific antibodies that are the same because they are made by identical immune cells that are all clones of a unique parent cell....

, are deposited on a chip surface (glass or silicon) in a miniature array. This format is often also referred to as a microarray (a more general term for chip based biological measurement devices).

Types of chips

There are several types of protein chips, the most common being glass slide chips and nano-well arrays.

Production of protein arrays

The production process depends on the type of protein chip.

Protein–Protein array: The proteins can be externally synthesised, purified and attached to the array. Alternatively they can be synthesised in-situ and directly attached to the array.
The proteins can be synthesised through biosynthesis

Protein biosynthesis

Protein biosynthesis is the process in which cells build or manufacture proteins. The term is sometimes used to refer only to protein translation but more often it refers to a multi-step process, beginning with amino acid synthesis and transcription of nuclear DNA into messenger RNA, which is then...

, cell-free DNA expression

Cell-free protein array

Cell-free protein array technology produces protein microarrays by performing in vitro synthesis of the target proteins from their DNA templates. This method of synthesizing protein microarrays overcomes the many obstacles and challenges faced by traditional methods of protein array production that...

or chemical synthesis

Peptide synthesis

In organic chemistry, peptide synthesis is the production of peptides, which are organic compounds in which multiple amino acids are linked via amide bonds which are also known as peptide bonds...

. In-situ synthesis is possible with the latter two. With cell-free DNA expression, proteins are attached to the support right after their production. Peptides chemically procured by solid phase peptide synthesis are already attached to the support. Selective deprotection is carried out through lithographic methods or by the so-called SPOT-synthesis.

DNA-Protein array: Double-stranded DNA (the exact binding sequence of the protein)is attached/ spotted on the array.

Artifacts to avoid

1) To avoid variability in results, use a very efficient lysis buffer and maintain consistent sample processing conditions;
2) Many antibodies don't work well as capture reagents, even if they do work well in western blotting and other denaturing conditions. Some antibodies often bind poorly to intact proteins in a cell extract;
3) Different proteins like different solution conditions, so if you do not see binding it doesn't mean that there is no binding between the two partners in physiological conditions;
4) Adjust the solute conditions to avoid non-specific association: change salt concentration, pH, add 1% alignate;
5) on the array's surface the conjugated protein should be in the right conformation (i.e., folded, NOT denatured), anchored by the same amino acid (in the same orientation), and be kept away from the surface by a linker to avoid steric hindrance.

Types of capture molecules

Capture molecules used are most commonly antibodies; however, antigens are used in applications where antibodies are detected in serum. More recently there has been a push towards other types of capture molecules which are more similar in their nature such as peptides or aptamers. Antibodies have several problems including the fact that there are no antibodies for most proteins and also problems with specificity in some commercial antibody preparations. Nevertheless, antibodies still represent the most well-characterized and effective protein capture agent for microarrays. Recently, nucleic acids, receptors, enzymes, and proteins have been spotted onto chips and used as capture molecules. This allows a vast variety of experiments to be conducted on protein–protein interactions, and all other protein binding substrates.

Detection methods

Although protein microarrays may use similar detection methods as DNA Microarrays, a problem is that protein concentrations in a biological sample may be many orders of magnitude different from that for mRNAs. Therefore, protein chip detection methods must have a much larger range of detection.

The preferred method of detection currently is fluorescence

Fluorescence

Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. It is a form of luminescence. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation...

detection. The fluorescent detection method is compatible with standard microarray scanners, the spots on the resulting image can be quantified by commonly used microarray quantification software packages. However, some minor alterations to the analysis software may be needed. Other common detection methods include colorimetric techniques based on silver-precipitation, chemiluminescent and label free Surface Plasmon Resonance.

Software/source code

Many software Packages are available to analyse methylation data, many of these are also freely available.

External links

Protein Array Resource Page on functionalgenomics.org.uk
'Self Assembly Nature's Way to Do It' Freeview Video of a Royal Institution Discourse by Kuniaki Nagayama, Tokyo University supplied by the Vega Science Trust.
Principles of Protein Microarrays; Mark Schena
Antigen Microarrays for Serodiagnosis of Infectious Diseases
Biomarker Discovery using Protein Arrays
Feature article about protein microarrays from the December 1, 2007 issue of Analytical Chemistry
Assays with Protein Arrays

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