Electrophoretic Mobility Shift Assay 1

Electrophoretic
mobility shift Assay
Group no. 15
16604 Fatima Tahir
16630 Iqra akbar
16638 Hajra Illayas
Introduction
• The electrophoretic mobility shift assay is a
powerful technique for detecting specific-binding
of nucleic acid-protein complexes.
• Over the past 30 years, EMSA has been the “go
to assay” to investigate the qualitative interactions
between nucleic acids (DNA or RNA) and nucleic-
acid binding proteins.
• It is rapid and sensitive method to detect protein
complex with the nucleic acid.
What is EMSA?
It is also known as Gel shift assay or gel mobility shift assay,
band shift assay, or gel retardation assay.
It is technique to detect weather protein is attached with

DNA or RNA or not. As we know, there are many cellular
processes where a protein needs to interact with the DNA or
RNA like in transcription and translation.
Originally utilized broadly in the investigation of sequence-

specific DNA-binding proteins such as transcription factors,
EMSA has been additionally developed to explore DNA-
protein interactions, RNA- protein interactions.
EMSA is a sensitive method, using radioisotopes to label

nucleic acids and autoradiography, it is possible to use very
low concentrations and small sample volumes.
Principle of EMSA
• The purified protein and the cell crude extract are usually incubated
with the 32P isotope-labeled DNA or RNA probe, and the complex
and the unbound probe are isolated on the non-denaturing
polypropylene gel electrophoresis.
A mobility shift assay is The speed at which

electrophoretic separation different molecules
of a protein-DNA or move through the gel
protein-RNA mixture on a is determined by their
polyacrylamide or agrose size, charge, and
gel for a short period. sometime by shape.
How it Works?
Steps involved in the working of EMSA:
• Preparation of cell protein extract.
• Prepare radioactively
labelled DNA.
• Binding reaction.
• Non-denaturing gel
electrophoresis.
• Detection of outcome.
Working of EMSA
• The protein (P) binding to the unique site on a
DNA (D), will form a complex PD; in equilibrium
with the free components.
When there is a strong interaction between proteins and

DNA ka ≥ Kd and a distinct band is observed.
However, due to dissociation occurs during

electrophoresis, a faint smear a faint smear will also show
between two bands.
If single DNA molecule has multiple binding sites for an

individual protein, there will be multiple complexes
formed, and we observe many bands.
Protocol or steps we follow in EMSA
1- Making labeled DNA:

It has three ways:
1-
2-
• Biotinylation:
2-Preparation of Gel:
Ensure the gel plate, spacer and comb are clean.
Remove all fingerprints through glass plate by
methanol and dry all components before use.
Prepare polymerization mixture.
Pour the mixture into the glass plate and make sure
to avoid bubble formation. Insert the comb
immediately.
3- Pre-electrophoresis:
• Remove comb and bottom spacer
from gel and mount the gel in the
vertical electrophoresis apparatus.
• Place glycerol and dye solution in
each well of the gel and conduct
pre-electrophoresis.
• This is done to just to see of the
gel has formed properly, rununig
the gel one it does not breakdown.

4- Sample preparation and equilibration:
 Prepare sample and equilibrate for 30 min at 20 + 1 ° C.
This can be carried out while the gel is undergoing pre-
electrophoresis.
5- Electrophoresis:
 Detection of electrophoretic bands on the basis of
radioactivity of samples.
 At the end, remove the gel and plate assembly from the
electrophoresis device and dry it.
6- Autoradiograph:
 To visualize gel bands.
 In a lab with dim light (for phosphorus screen) place film
or phosphorus in an exposure casset.
• Multiple protein binding:
• Super shift:
Control Study understudy and result
How and why titration is done?
• As we know that, we are going to have DNA
interacting with protein and for that to be
happen we need to have enough DNA and
protein in the solution.
Applications
It is used to study
conformational Analysis of cellular
changes in DNA . For extracts for the It proves useful
example a EMSA 2-D presence of certain for stoichiometric
variant with electron DNA-binding analysis. Number
microscopy is used proteins. Bound and of protein that
for the free DNA are bind per DNA
characterization of separated using fragment
conformation. EMSA.
It is also used too study wether transcription factor is attached to the DNA or not or if
transcription factor attached is our targeted or not and their activity.
Advantages of EMSA
It can be used with wide range of nucleic acid, sizes, structure
as well as wide range of proteins.
It is relatively simple method to perform. It is also used for

qualitative analysis.
It proves useful in detection of protein that binds to DNA, and

cause difficulty in migration of fragment on gel.
Highly sensitive method and usually of Low cost.

No special equipment needed.
In this technique, it is possible to use both crude protein extracts

and purified recombinant proteins.
Drawbacks of EMSA:
EMSA doesn’t provide a straight forward measure of
the weights of the proteins as mobility is influenced by
several other methods.
Not an appropriate method for kinetic studies.
Dissociation is also one of the drawback of EMSA. It

occurs during electrophoresis thus prevents detection.
Interacting biomolecules must have different

electrophoretic mobilities.
Conclusion:
Used for various

purposes such as
quantifying
interactions
between DNA and Electrophoretic mobility shift
proteins assay (EMSA) is most widely
used method for the
detection of protein-DNA
interactions.

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Electrophoretic

mobility shift Assay

It is technique to detect weather protein is attached with

Originally utilized broadly in the investigation of sequence-

EMSA is a sensitive method, using radioisotopes to label

A mobility shift assay is The speed at which

When there is a strong interaction between proteins and

However, due to dissociation occurs during

If single DNA molecule has multiple binding sites for an

1- Making labeled DNA:

It is relatively simple method to perform. It is also used for

It proves useful in detection of protein that binds to DNA, and

Highly sensitive method and usually of Low cost.

In this technique, it is possible to use both crude protein extracts

Not an appropriate method for kinetic studies.

Dissociation is also one of the drawback of EMSA. It

Interacting biomolecules must have different

Used for various

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