Lesson 88
Protein Microarray
Protein Microarray
A protein microarray (or protein chip) is a high-throughput method used to track
the interactions and activities of proteins, and to determine their function, and
determining function on a large scale. Its main advantage lies in the fact that large
numbers of proteins can be tracked in parallel. The chip consists of a support
surface such as a glass slide, nitrocellulose membrane, bead, or microtitre plate, to
which an array of capture proteins is bound. Probe molecules, typically labeled
with a fluorescent dye, are added to the array. Any reaction between the probe and
the immobilised protein emits a fluorescent signal that is read by a laser scanner.
Protein Microarray
Protein microarrays are rapid, automated, economical, and highly
sensitive, consuming small quantities of samples and reagents. The
high-throughput technology behind the protein microarray was
relatively easy to develop since it is based on the technology developed
for DNA microarrays, which have become the most widely
used microarrays
Types of arrays
There are three types of protein microarrays that are currently used to study the
biochemical activities of proteins. Analytical microarrays are also known as capture
arrays. In this technique, a library of antibodies, aptamers or affibodies is arrayed
on the support surface. These are used as capture molecules since each binds
specifically to a particular protein. The array is probed with a complex protein
solution such as a cell lysate. Analysis of the resulting binding reactions using
various detection systems can provide information about expression levels of
particular proteins in the sample as well as measurements of binding affinities and
specificities. This type of microarray is especially useful in comparing protein
expression in different solution
RPPA
Reverse phase protein microarray (RPPA) involve complex samples, such as tissue
lysates. Cells are isolated from various tissues of interest and are lysed. The lysate is
arrayed onto the microarray and probed with antibodies against the target protein
of interest. These antibodies are typically detected with chemiluminescent,
fluorescent or colorimetric assays. Reference peptides are printed on the slides to
allow for protein quantification of the sample lysates. RPAs allow for the
determination of the presence of altered proteins or other agents that may be the
result of disease. Specifically, post-translational modifications, which are typically
altered as a result of disease can be detected using RPAs
Functional protein microarrays
Functional protein microarrays (also known as target protein arrays) are
constructed by immobilising large numbers of purified proteins and are
used to identify protein–protein, protein–DNA, protein–RNA, protein–
phospholipid, and protein–small-molecule interactions, to assay
enzymatic activity and to detect antibodies and demonstrate their
specificity. They differ from analytical arrays in that functional protein
arrays are composed of arrays containing full-length functional proteins
or protein domains. These protein chips are used to study the
biochemical activities of the entire proteome in a single experiment.
Functional protein microarrays
• The key element in any functional protein microarray-based assay is
the arrayed proteins must retain their native structure, such that
meaningful functional interactions can take place on the array
surface. The advantages of controlling the precise mode of surface
attachment through use of an appropriate affinity tag are that the
immobilised proteins will have a homogeneous orientation resulting
in a higher specific activity and higher signal-to-noise ratio in assays,
with less interference from non-specific interactions
Sources:
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