1. Protein Microarray
A high -throughput screening method
Master’s Seminar (BMB600)
Allena Priyadarsini Sahoo
M.Sc. Agriculture Biotechnology
Id No. 57223
Molecular Biology and Genetic Engineering,
College of Basic Sciences and Humanities
G.B. Pant University of Agriculture and Technology

2. What is microarray ?
 Arrays are solid support upon which a
collection of gene specific probe 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 2 type of Arrays
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 DNA micro-Array (An indirect method for proteomic)
 Protein micro-Array (A direct method for proteomic)

3.  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
Motivation for development-
 Developed due to the limitations of using DNA microarrays for
determining gene expression levels in proteomics.
 Additionally post-translational modifications, which are often
critical for determining protein function, are not visible on DNA
microarrays.

4.  Protein chip consists of a support surface
such as glass slide, nitrocellulose ,bead or
micro-titer plate to which array of capture
proteins is bound.
 Probe molecules typically labelled with a
fluorescent dye are added to the array.
 A reaction between the probe and the
immobilized protein emits a fluorescent
signal and that is read by a laser scanner
Principle-

5. Principle-

6. The probe on chip
A variety of material can be immobilize on the protein chip based on the
specific requirement .This include
1.Antibodies
2.Antigens
3.Aptamers(nucleic acid based ligand)
4.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.)
5.Full length protein or , their domain

7. The sources of probes on chip-
1. Cell lysates-if you are interested in full length
protein) by using different chromatographic
techniques like affinity chromatography, ion
exchange chromatography).
2. Recombinant DNA Technology-

8. 3.Synthetic peptides- The established method for the production of synthetic
peptides in the lab is known as solid-phase peptide synthesis (SPPS).

9. 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
1. Cell extract
2. An energy source( phosphoenol pyruvate ,acetyl
phosphate,and creatine phosphate)
3. Supply of amino acids
4. Cofactors ,such as mg2+(for keeping ribosome intact)
5. The DNA with the desired gene.
6. The necessary cell machinery including ribosomes ,
aminoacyl-trna synthetases ,translation initiation and
elongation factor, etc

10. Nucleic Acid programmable protein Array(NAPPA)-
Avidin is a tetrameric biotin binding protein. Biotin is a water soluble vitamin.

11. Drawbacks of NAPPA-
The main drawback of this method is the extra and tedious preparation steps
at the beginning of the process
Resulting protein array is not pure because the proteins are co-localized
with their DNA template and capture antibodies.
Protein in situ array(PISA)
Unlike , NAPPA ,PISA completely bypasses DNA immobilization as the
DNA template is added as a free molecule in the , reaction mixture ,so in
the end the DNA will not be present in the well of the chip.

12. Detection system -

13. Types of protein Microarray-
• Analytical microarrays are also known as capture arrays,uses a
library of antibodies , Aptamers or Affibodies 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. This type of microarray is especially useful in
comparing protein expression in different solutions.
• For instance the response of the cells to a particular factor can be
identified by comparing the lysates of cells treated with specific
substances or grown under certain conditions with the lysates of
control cells. Another application is in the identification and
profiling of diseased tissues.

14. Analytical microarray-

15. Functional microarray-
• also known as( target protein arrays ) are constructed by
immobilizing large numbers of purified proteins and are used to
identify protein-protein , protein-DNA ,protein-RNA and protein
small molecule interaction , 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
or protein domains .
• These protein chips are used to study the biochemical activities
of the entire proteome in a single experiment.

17. Application of protein
Microarray-
• Functional protein microarrays provide a flexible platform that can
characterize a wide range of biochemical properties of spotted
proteins.
• To date,these assay have been successfully developed to detect
various types of protein-binding properties ,such as protein-protein,
protein-DNA , protein-RNA , protein-drug and identify substrate of
various classes of enzymes ,such as protein kinase , ligases.
• Biomarkers are a crucial tool in the expeditious detection of
infection or diagnosis of autoimmune disease.
• Protein post –translational modification are one of the most
important mechanisms to directly regulate protein activity . Among
the hundreds of PTMS identified ,in addition to glycosylation,
enzyme-dependent , reversible protein de(phosphorylation) and
de(acetylation) are perhaps the most well studied.

18. • A new trend in the field of host –pathogen interactions is to
use host protein microarrays to survey relationship between
a pathogenic factor of interest and the host proteome.this
idea is particularly suited for studying host-virus interaction
because ,after entering the host cells ,the viral genome and
proteins are in direct physical contact with the host
compoments ,where by a pathogen can hajack/exploit the
host pathways and machineries for its own replication.

19. Challenges-
Despite the considerable investments made by several companies ,
protein chip has yet to flood the market .Manufacturers have found that
the proteins are actually quite difficult to handle . A protein chip
requires a lot more steps in its creation than does a DNA chip.
Challenges include
Finding a surface and a method of attachment that allows the proteins
to maintain their secondary/tertiary structure and thus their biological
activity and their interaction with other molecules.
Producing an array with a long shelf life so that proteins on the chip do
not denature over a short time.

20. • Identifying and isolating antibodies or other capture molecules
against every protein in the human genome.
• Quantifying the levels of bound protein while assuring sensitivity
and avoiding background noise.
• Extracting the detected protein from the chip in order to further
analyse it.
• Reducing non-specific binding by the capture agents.
• The capacity of the chip must be sufficient to allow as complete a
representation of the proteome to be visualized as possible ; abudant
protein overwhelm the detection of less abundant proteins such as
signalling molecules and receptors ,which are generally of more
therapeutic interest.