This document describes an electrophoretic mobility shift assay (EMSA) presentation. EMSA is a technique used to study interactions between proteins and DNA. It works by detecting a reduction in electrophoretic mobility of DNA when bound to a protein through gel electrophoresis. The presentation aims to describe the basic principles of EMSA, highlight its methods, and discuss applications such as determining binding affinities and studying conformational changes in DNA upon protein binding.

1. BGM3009 Presentation
On
Electrophoretic Mobility Shift Assay
(EMSA):
A Method for Analysing Protein-DNA Interactions
Presenters:
Kate Wisdom, Songo Lolomari, Nicholas Leach & Abhay Jethwani
Aims and Objectives:
• To describe the basic principles behind EMSA
• To highlight methods used in EMSA
• To discuss the applications of EMSA including
– Kd evaluation &
– Conformation changes in DNA upon protein binding.

2. Definition and Basic Principle
•Technique used to study interactions
between proteins and DNA.
•Simple, efficient and sensitive
technique
•DNA moves through the gel faster
when not bound to protein
•A reduction in electrophoretic mobility
shows that a complex is formed
between DNA and protein
•Can be used to identify DNA-binding
proteins present in a nuclear cell
extract. For example, transcription
factors.
Fig. Source: http://universe-review.ca/R11-16-DNAsequencing.htm

3. Methods
5 basic steps are in
conventional EMSA protocol
–Preparation of purified or
crude protein sample
–Preparation of nucleic acid
–Binding reactions
–Non-denaturing gel
electrophoresis
–Detection of the outcome
EMSA Variant: Supershift
assay
Yang, V.W. (1998), JN, 128(11), pp. 2045-2051

4. EMSA Applications (I)
DNA
Protein
Complex
Kd EVALUATION
• Kd can be calculated as the concentration
when 50% of receptors are occupied
• For a protein-DNA reaction, the receptor
(binding site) is on the DNA
• Hence, Kd is gotten when 50% of DNA is
bound

5. EMSA Applications (I)
Using EMSA to determine kd
1
• A fixed concentration of DNA is
titrated with excess protein
2
• Bound and free DNA are separated
using EMSA
3
• Measure density of bands, Kd=protein
concentration when 50% of DNA is
free
• True kd can only be measured when stable protein-DNA
complexes are formed for over a long period (sub
nanomolar kd range)
• Apparent kd measured often measured instead
• Zone interference electrophoresis usually used to
measure less stable complexes (10¯⁶ to 10‾ ⁴ kd range)

6. Kd evaluation example
• Study of the kd for binding between
BV 04-01 and different ssDNA
homopolymers
• BV 04-01= anti-DNA antibody
implicated in SLE in mice
• EMSA done to confirm results
obtained with other methods
• Showed oligo (T) had best affinity
with a kd of ~4.4nM, similar to that
obtained using other methods
• In addition, showed that antibody
also had a high affinity for oligo (dG)
(140nM).
• Helped in the understanding of
affinity of the anti-DNA antibody
for ssDNA.
Graph of % free oligo (dG) against
protein concentration, used to measure
Kd. Each point on plot is an average of 3
binding titrations(Stevens et al, 1994).
EMSA Applications (I)

7. EMSA Applications (II)
STUDY OF CONFORMATIONAL CHANGE IN DNA
The effect of DNA bending on migration in the gel:
• effect of the bend position on gel mobility
The position of the bend on the DNA influences:
• end-to-end distance
• cross sectional area
position of the bend (small circle) on
each DNA molecule A-G
Adapted from Lane et. Al. Microbiol. Rev 1992.
SHIFT
a)
b)
c)
Bend at centre
The migration is lower when the bend is
closer to the centre of the DNA
molecule

8. Example of a circular permutation
polyacrylamide gel retardation assay (1)
• Studies the bending of DNA by
p53DBD protein (human p53) by
binding to several natural occurring
response elements
• Oligonucleotides used contain a 20bp
response element within a 30mer
oligonucleotide.
-cloned into DNA vector pBend3
using XbaI and SalI restriction
sites
• Recombinant plasmids are digested
by various restriction enzymes
-p53 response element at
increasing distances across the
DNA molecule.
• Fragments are 32P-radiolabelled.
Adapted from Nagaich et al. JBC, 1997
EMSA Applications (II)

9. Example of a circular permutation
polyacrylamide gel retardation assay (2)
• EMSA is used for the p53DBD-DNA
complexes.
• Electrophoretic mobility of the
complex is dependent upon the
position of p53DBD on the DNA
c) d)
Adapted from Nagaich et al. JBC, 1997
Restriction enzyme Restriction enzyme
• Bending angles can be determined:
μM= mobility of complex when binding
site is at the middle of DNA
μE=mobility of complex when binding site
is at the end of DNA
α=angle of induced bend
k= coeffecient
EMSA Applications (II)

10. Advantages and Limitations
Advantages
• It is a simple method to
perform but yet is robust
enough to include a wide range
of conditions.
• Highly sensitive method .
Assays could be performed
with small nucleic acid
concentrations and small
sample volumes.
• EMSA could also be used with
a wide range of nucleic acid
sizes and structures as well as
wide range of proteins.
• Finally, it is possible to use
both crude protein extracts
and purified recombinant
proteins.
Limitations
• Dissociation is one of the
drawbacks of EMSA. It occurs
during electrophoresis thus
prevents detection.
• EMSA doesn’t provide
information on the nucleic acid
sequence the proteins are bound
to.
• Not an appropriate method for
Kinetic studies.
• Does not provide a
straightforward measure of the
weights of the proteins as
mobility is influenced by several
other factors.

11. Conclusion
• Electrophoretic Mobility Shift Assay (EMSA) is the most
widely used method for the detection of protein-DNA
interactions.
• Works on the observation that protein-bound DNA migrate
slowly as compared to free DNA when subjected through
electrophoresis through a non-denaturing gel.
• Used for various purposes such as quantifying interactions
between proteins and DNA, determination of binding
affinities but most importantly in the characterization of
Transcription Factors.
• There are several alternatives to EMSA which include Foot
printing, Yeast hybrid systems, etc.

12. References
• Alves, C. and C. Cunha, Gel Electrophoresis - Advanced Techniques. Electrophoretic
Mobility Shift Assay: Analyzing Protein - Nucleic Acid Interactions, ed. M. Sameh.
2012: InTech. 500.
• Hellman, L.M. and M.G. Fried, Electrophoretic mobility shift assay (EMSA) for
detecting protein-nucleic acid interactions. Nat. Protocols, 2007. 2(8): p. 1849-1861.
• Lane, D., Prentki, P. and Chandler, M. (1992) 'Use of gel retardation to analyze
protein-nucleic acid interactions', Microbiological Reviews, 56(4), pp. 509-528.
• Nagaich, A.K., Appella, E. and Harrington, R.E. (1997) 'DNA Bending Is Essential for
the Site-specific Recognition of DNA Response Elements by the DNA Binding
Domain of the Tumor Suppressor Protein p53', Journal of Biological Chemistry,
272(23), pp. 14842-14849.