ASSIGNMENT-4

GENOMICS & PROTEOMICS

[KBT-071]

Title: PCR-Directed Protein In Situ Arrays

Submitted to: Submitted by:

Prof. Vaishnavi Garg Monisha Singh

Department of Biotechnology 200010540005

AEC IV year
Polymerase Chain Reaction (PCR) is a molecular biology technique commonly used to
amplify and analyze DNA. It is not typically used for amplifying proteins directly, as
proteins are generally more complex molecules compared to DNA. However, there are
methods to study proteins in situ (meaning within their natural environment or location)

using various techniques.

"PCR-Directed Protein In Situ Arrays" might imply a methodology

where PCR is used to guide or direct the analysis of proteins in their original context.
This could involve amplifying specific DNA sequences related to proteins of interest and
then using these amplified sequences to study the corresponding proteins in their
natural cellular or tissue environment.

Protein Array

Protein array technology involves the use of miniature arrays or microarrays to

simultaneously study the presence, abundance, and interactions of multiple proteins
within a biological sample. These arrays are often designed on solid surfaces, such as
glass slides or membranes, with each spot containing a unique protein or peptide.

Applications:

 protein-protein interactions
 protein binding, protein expression profiling
 Antibody characterization.

Types of Protein Array

1. Forward Protein Array

Analytical Protein Arrays:

Analytical protein arrays are designed to analyze various characteristics of proteins, such
as their interactions, modifications, and expression levels. Example: Protein Interaction
Arrays
Applications:

 Protein-Protein Interactions

 Post-Translational Modifications

Functional Protein Arrays:

Functional protein arrays focus on assessing the biological activity of proteins. This can
include enzymatic activity, pathway activation, or other functional aspects. Example:
Enzyme Activity Profiling Array

Applications:

 Enzyme Activity Profiling

 Pathway Activation Studies

Reverse Phase Protein Arrays (RPPA)
Reverse Phase Protein Arrays (RPPA) are a high-throughput technology used to

quantitatively measure the levels of specific proteins in a large number of

biological samples. Unlike traditional protein arrays where antibodies are
immobilized on the array surface, RPPA involves spotting patient samples or cell
lysates onto the array, and these samples are then probed with specific
antibodies.

Applications:

1. Protein Expression Profiling:

 RPPA allows researchers to simultaneously measure the expression levels

of multiple proteins across a large number of samples.

 Example: Investigating the expression of key signaling proteins in cancer

samples to identify potential biomarkers or therapeutic targets.

2. Pathway Analysis:

 By measuring the activation status of proteins within specific signaling

pathways, RPPA can provide insights into pathway dysregulation.

 Example: Studying the activation of the PI3K/Akt/mTOR pathway in

response to different treatments in cancer cells.

3. Biomarker Discovery:

 RPPA is used to identify proteins that are differentially expressed in

disease states, aiding in the discovery of diagnostic or prognostic
biomarkers.

 Example: Identifying proteins associated with drug resistance in tumor

samples to guide personalized treatment strategies.

Advantages:
1. High Throughput: RPPA allows the analysis of numerous samples simultaneously,
making it suitable for large-scale studies.

2. Quantitative Analysis: Provides quantitative information about protein levels,

allowing for precise comparisons between samples.

3. Sample Efficiency: Requires small amounts of sample, making it suitable for

studies with limited biological material.

4. Complementary to Genomic Data: Offers complementary information to

genomic data by assessing protein expression and activation directly.

Fig. Reverse Phase Protein Arrays (RPPA)

Protein array in situ techniques

PISA

PISA (protein in situ array) completely bypasses DNA

immobilization as the DNA template is added as a free molecule

in the reaction mixture. In 2006, another group refined and

miniaturized this method by using multiple spotting techniques

to spot the DNA template and cell-free transcription and

translation mixture on a high-density protein microarray with up

to 13,000 spots. This was made possible by the automated

system used to accurately and sequentially supplies the reagents

for the transcription/translation reaction occurs in a small, sub-

nanolitre droplet.
NAPPA

NAPPA uses DNA template that has already been immobilized

onto the same protein capture surface. The DNA template

is biotinylated and is bound to avidin that is pre-coated onto

the protein capture surface. Newly synthesized proteins which

are tagged with GST are then immobilized next to the template
DNA by binding to the adjacent polyclonal anti-GST capture

antibody that is also pre-coated onto the capture surface.

DAPA

DNA array to protein array (DAPA) is a method developed in

2007 to repeatedly produce protein arrays by ‘printing’ them

from a single DNA template array.

It starts with the spotting and immobilization of an array of

DNA templates onto a glass slide. The slide is then assembled

face-to-face with a second slide pre-coated with a protein-

capturing reagent, and a membrane soaked with cell extract is

placed between the two slides for transcription and translation

to take place. The newly synthesized his-tagged proteins are

then immobilized onto the slide to form the array. In the

publication in 18 of 20 replications a protein microarray copy

could be generated. Potentially the process can be repeated as

often as needed, as long as the DNA is unharmed by DNAses,

degradation or mechanical abrasion.
Advantages

Many of the advantages of cell-free protein array technology

address the limitations of cell-based expression system used in
traditional methods of protein microarray production.

1. Rapid and cost-effective

The method avoids DNA cloning (with the exception of NAPPA)

and can quickly convert genetic information into functional

proteins by using PCR DNA. The reduced steps in production

and the ability to miniaturize the system saves on reagent

consumption and cuts production costs.

2. Improves protein availability

Many proteins, including antibodies, are difficult to express in

host cells due to problems with insolubility, disulfide bonds or

host cell toxicity. Cell-free protein array makes many of such

proteins available for use in protein microarrays.

3. Enables long term storage

Unlike DNA, which is a highly stable molecule, proteins are a

heterogeneous class of molecules with different stability and

physiochemical properties. Maintaining the proteins’ folding and

function in an immobilized state over long periods of storage is

a major challenge for protein microarrays. Cell-free methods

provide the option to quickly obtaining protein microarrays on
 demand, thus eliminating any problems associated with long-

term storage.

4. Flexible

The method is amenable to a range of different templates: PCR

products, plasmids and mRNA. Additional components can be

included during synthesis to adjust the environment for protein

folding, disulfide bond formation, modification or protein

activity.

Limitation

 Tedious preparation steps at the beginning of the

process

 Post-translational modification of proteins in

proteins generated by cell-free protein synthesis is

still limited compared to the traditional methods, and

may not be as biologically relevant.

Applications

 Protein interactions: To screen for protein–protein

interactions and protein interactions with other molecules such

as metabolites, lipids, DNA and small molecules.
 Enzyme inhibition assay: for high throughput drug candidate

screening and to discover novel enzymes for use

in biotechnology.
 Screening antibody specificity.