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Protein Microarray Naveed Up Mushtaq

Protein microarrays allow the high-throughput analysis of protein interactions and functions. They contain small amounts of purified proteins arranged in a dense format on a surface, enabling the simultaneous determination of various analytes from small sample amounts. There are three main types: analytical microarrays use antibodies to detect proteins in samples, functional microarrays immobilize proteins to study interactions, and reverse phase microarrays profile protein expression by spotting cell lysates. Protein microarrays have applications in diagnostics, proteomics, analyzing protein functions, characterizing antibodies, and developing new treatments.

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141 views16 pages

Protein Microarray Naveed Up Mushtaq

Protein microarrays allow the high-throughput analysis of protein interactions and functions. They contain small amounts of purified proteins arranged in a dense format on a surface, enabling the simultaneous determination of various analytes from small sample amounts. There are three main types: analytical microarrays use antibodies to detect proteins in samples, functional microarrays immobilize proteins to study interactions, and reverse phase microarrays profile protein expression by spotting cell lysates. Protein microarrays have applications in diagnostics, proteomics, analyzing protein functions, characterizing antibodies, and developing new treatments.

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Angumani
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PROTEIN

MICROARRAY
NAVEED UP MUSHTAQ
Protein microarray
A protein microarray (protein chip)is a high
throughput method used to track the
interactions & activities of proteins,& to
determine their functions,& determining
function on a large scale.
Protein microarrays,are miniaturised and
parallel assay systems that contain small
amounts of purified proteins in a high density
format.
They allow simultaneous determination of a
great variety of analytes from small amounts of
samples within a single experiment.
This technology can complement other
techniques,such as mass spectrometry &
yeast two-hybrid assays,to identify
thousands of protein-protein
interactions.Protein arrays can be screened
for their ability to bind other proteins in a
complex,receptors,antibodies;lipids;enzym
es;pepyides;harmones;specific DNA
sequence.
Preparation of protein microarray
Protein microarrays are typically
prepared by immobilizing proteins onto a
microscope slide using a standard contact
spotter or non contact microarray.
Different methods of arraying the proteins:-
Robotic method
Ink jetting method
Piezoelectric spotting
Photoliyhography.
In these methods,robotic is contact microarray
method while the other three are non contact
microarray methods.
A variety of slide surfaces can be used.popular
types include aldehyde & epoxy-derivatized glass
surfaces for random attachment through
amines,nitrocellulose or gel coated slides and nickel
coated slides for affinity attachment of HIS 6-tagged
proteins.
After proteins are immobilised on the slides,they
can be probed for a variety of
functions/activities.Typically the probe molecules are
labelled with fluoresent dye,so that when the probe
binds to the protein it results in a fluoresent
signal.Finally the resulting signals are usually
measured by detecting these labels.
TYPES OS PROTEIN MICROARRAYS
There are three types of protein microarrays
that are currently used to study the
biochemical activities of proteins.
Analytical protein microarrays
Functional protein microarrays
Reverse phase protein microarrays.
1. Analytical microarrays:-
Analytical microarrays(or antibody microarrays)
have antibodies arrays on solid surface,and are used
to detect proteins in biological samples.Often a
second is used to detect a protein that is captured by
the antibody attached to the solid phase,in a principle
similar to that of sand wich immunoassay, in which
the first antibody is spotted on the array and then a
captured antigen on the chip is detected with a
second antibody that recognises a different part of
antigen.
2.FUNCTIONAL PROTEIN MICROARRAYS:-
Also known as target protein array.With functional
protein microarrays purified recombinant protein are
immobilised onto the solid phase.
Functional protein microarrays have recently been
applied to many aspects of discovery based
biology,including protein-protein,protein-
lipid,protein-DNA,protein-drug,&protein –peptide
iinteractions.
These can be used to identify enzyme substrates.
These can also be used to detect antibodies in a
biological specimen to profile an immune response.
3.REVERSE PHASE PROTEIN MICROARRAY:-
Involves complex samples, such as tissue lysates.cells
are isolated from various tissues of interest and
lysed.The lysate is arranged onto the microarray &
probed with antibodies against the target protein of
interest.These antibodies are typically detected with
chemiluminescent,fluoresent or colorimetric assays.
RPAs allow for the determination of the presence of
altered proteins or other agents that may be the
result of disease.
Specifically,post translationl modifications,which
are typically altered as a result of disease can be
detected using RPAs.
APPLICATIONS:-
There are five major areas where protein arrays are
being applied:diagnostics,proteomics,protein
functional analysis,antibody characterisation &
treatment.
Diagnostics involves the detection of antigens &
antibodies in blood samples;to discover new disease
biomarkers;the monitoring of disease states &
responses to therapy in personalised medicine;the
monitoring of environment & food.
Proteomics pertains to protein expression profilling
i.e;which proteins are expressed in the lysate of a part
of cell.
Protein functional analysis is the identification
of protein-protein interactions,protein-
phospholipid interactions,small molecule
targets,enzymatic substrates & receptor ligands.
Antibody characterization is characterizing
cross reactivity,specificity & mapping epitopes.
Treatment development involves the
development of antigen-specific therapies for
autoimunity,cancer & allergies;the identification
of small molecule targets that could potentially
be used as new drugs.
THANK YOU

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