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GEL RETARDATION ANALYSIS


GEL RETARDATION ANALYSIS (EMSA – Electrophoretic Mobility Shift Assay)


Introduction


Gel retardation analysis, also known as Electrophoretic Mobility Shift Assay (EMSA), is a widely used in vitro technique for studying DNA–protein and RNA–protein interactions.
The method is based on the observation that a DNA–protein complex migrates more slowly than free DNA during non-denaturing gel electrophoresis, resulting in a mobility shift or “retardation”.
EMSA is extensively used to study transcription factor binding, regulatory DNA elements, and binding specificity.


Definition


Gel retardation analysis (EMSA) is a technique used to detect and analyze binding interactions between nucleic acids and proteins by observing the reduced electrophoretic mobility of nucleic acid–protein complexes compared to free nucleic acids.

Principle

A labeled DNA or RNA probe is incubated with a specific binding protein.
When binding occurs, a nucleic acid–protein complex is formed.
This complex has a larger size and altered charge, causing it to migrate slower than free nucleic acid in a native polyacrylamide gel.
The shift in position of the band indicates binding interaction.

Key principle:

Bound nucleic acid moves slower than free nucleic acid → gel retardation.
Requirements / Materials
DNA or RNA probe (labeled with radioisotope, fluorescence, or biotin)
DNA-binding or RNA-binding protein
Binding buffer (physiological conditions)
Competitor DNA (specific and non-specific)
Native polyacrylamide gel electrophoresis system
Detection system (autoradiography, fluorescence scanner, chemiluminescence)
Methodology


1. Preparation of Nucleic Acid Probe


A short double-stranded DNA fragment containing the suspected binding site is selected.
The probe is labeled at one end using:
Radioactive label (³²P)
Fluorescent dye
Biotin
2. Binding Reaction
Labeled probe is incubated with protein extract or purified protein.
Reaction is performed under native, non-denaturing conditions.
Binding buffer maintains optimal pH, ionic strength, and temperature.

3. Competition Assay (Optional but Important)
Specific competitor DNA (unlabeled but identical sequence) competes with labeled probe → confirms specificity.
Non-specific competitor DNA (e.g., poly dI-dC) reduces non-specific binding.

4. Supershift Assay (Optional)


Addition of a specific antibody against the DNA-binding protein.
Antibody-protein-DNA complex migrates even slower → supershift, confirming protein identity.

5. Gel Electrophoresis

Samples loaded onto native polyacrylamide gel.
Electrophoresis carried out without denaturants to preserve complexes.
6. Detection

After electrophoresis, gel is:
Exposed to X-ray film (autoradiography), or
Scanned for fluorescence/chemiluminescence.


Applications

Detection of DNA–protein interactions
Study of transcription factor binding
Analysis of promoter and enhancer elements
Determination of binding specificity and affinity
RNA–protein interaction studies
Drug screening for DNA-binding inhibitors


Advantages


Simple and sensitive technique
Requires small amounts of DNA and protein
Can detect specific and non-specific binding
Allows protein identification using supershift

Limitations


Does not provide exact nucleotide binding site
In vitro technique (may not reflect in vivo conditions)
Cannot determine protein molecular weight directly
Complex interpretation if multiple proteins bind



Conclusion


Gel retardation analysis (EMSA) is a powerful and widely used technique for detecting nucleic acid–protein interactions. Although it does not provide nucleotide-level resolution, it is invaluable for studying binding specificity, affinity, and regulatory proteins involved in gene expression.



50 MCQs – Gel Retardation Analysis (EMSA)


Basic Concepts
Gel retardation analysis is also known as:
A) Southern blotting
B) DNA footprinting
C) Electrophoretic Mobility Shift Assay (EMSA) ✅
D) Northern blotting
EMSA is used to study:
A) DNA replication
B) DNA–protein interactions ✅
C) Protein–protein interactions
D) DNA sequencing
The term “gel retardation” refers to:
A) DNA degradation
B) Slower migration of DNA–protein complex ✅
C) Faster migration of DNA
D) Gel polymerization
EMSA is mainly an:
A) In vivo technique
B) In vitro technique ✅
C) Clinical technique
D) Sequencing method
In EMSA, free DNA migrates:
A) Slower than DNA–protein complex
B) Faster than DNA–protein complex ✅
C) At same speed
D) Does not migrate
Principle
The mobility shift occurs due to:
A) DNA denaturation
B) Increase in size of complex ✅
C) DNA cleavage
D) Protein degradation
EMSA is performed under:
A) Denaturing conditions
B) Reducing conditions
C) Native conditions ✅
D) Alkaline conditions
The gel used in EMSA is:
A) Agarose gel
B) Denaturing PAGE
C) Native polyacrylamide gel ✅
D) SDS-PAGE
Binding between DNA and protein depends on:
A) Temperature
B) Ionic strength
C) pH
D) All of the above ✅
The probe used in EMSA is usually:
A) Single-stranded RNA
B) Double-stranded DNA containing binding site ✅
C) Protein fragment
D) Lipid molecule
Probe & Labeling
The probe in EMSA is labeled for:
A) Protein binding
B) Detection after electrophoresis ✅
C) DNA digestion
D) DNA methylation
Common radioactive label used in EMSA is:
A) ¹⁴C
B) ³²P ✅
C) ³H
D) ¹²C
Fluorescent labeling helps in:
A) Cleavage of DNA
B) Visualization without radioactivity ✅
C) Protein denaturation
D) DNA replication
Probe length in EMSA is generally:
A) Very long (>10 kb)
B) Short (20–30 bp) containing binding site ✅
C) Entire genome
D) Random DNA
Unlabeled probe is also known as:
A) Marker
B) Ladder
C) Competitor DNA ✅
D) Primer
Binding & Competition Assay
Specific competitor DNA is used to:
A) Enhance binding
B) Confirm binding specificity ✅
C) Denature protein
D) Label DNA
Non-specific competitor DNA is used to:
A) Increase binding
B) Reduce non-specific binding ✅
C) Digest DNA
D) Denature gel
Common non-specific competitor DNA is:
A) Poly dI–dC ✅
B) Plasmid DNA
C) RNA
D) cDNA
Disappearance of shifted band after adding specific competitor indicates:
A) Non-specific binding
B) DNA degradation
C) Specific DNA–protein interaction ✅
D) Protein denaturation
Increasing protein concentration generally results in:
A) No binding
B) Stronger shifted band ✅
C) DNA degradation
D) Faster migration
Supershift Assay
Supershift assay involves addition of:
A) Restriction enzyme
B) Antibody against DNA
C) Antibody against binding protein ✅
D) DNA polymerase
Supershift confirms:
A) DNA sequence
B) Protein identity in complex ✅
C) DNA length
D) Gel concentration
Supershifted band migrates:
A) Faster than shifted band
B) Same as free DNA
C) Slower than DNA–protein complex ✅
D) Off the gel
Absence of supershift suggests:
A) No protein present
B) Incorrect antibody used ✅
C) DNA is degraded
D) Gel failure
Supershift assay is mainly used for:
A) Protein purification
B) Identifying transcription factors ✅
C) DNA sequencing
D) RNA isolation
Detection & Interpretation
EMSA results are detected by:
A) PCR
B) Autoradiography or fluorescence scanning ✅
C) Western blotting
D) ELISA
Free DNA appears as:
A) Upper band
B) Lower band in gel ✅
C) Smear
D) No band
DNA–protein complex appears as:
A) Lower band
B) Smear
C) Shifted upper band ✅
D) No band
Multiple shifted bands indicate:
A) DNA degradation
B) Multiple proteins or complexes binding ✅
C) Gel melting
D) Poor labeling
Intensity of shifted band reflects:
A) DNA length
B) Protein concentration and affinity ✅
C) Gel thickness
D) Buffer pH
Applications
EMSA is commonly used to study:
A) DNA repair
B) Transcription factor binding ✅
C) Protein synthesis
D) RNA splicing
EMSA helps identify:
A) Promoters and enhancers ✅
B) Ribosomes
C) Introns only
D) Lipids
RNA EMSA is used to study:
A) DNA replication
B) RNA–protein interactions ✅
C) Translation only
D) DNA methylation
EMSA is useful in drug discovery to:
A) Sequence DNA
B) Screen DNA-binding inhibitors ✅
C) Purify proteins
D) Measure RNA levels
EMSA is preferred because it:
A) Is genome-wide
B) Is simple and sensitive ✅
C) Gives nucleotide sequence
D) Works only in vivo
Advantages & Limitations
Major advantage of EMSA is:
A) Identifies exact binding nucleotide
B) Simple and requires small sample amounts ✅
C) Works only for RNA
D) No controls required
EMSA cannot provide:
A) Evidence of binding
B) Binding specificity
C) Exact binding site on DNA ✅
D) Binding affinity
EMSA is less suitable when:
A) Protein binds weakly
B) Multiple proteins bind same site
C) In vivo confirmation is required ✅
D) DNA fragment is short
Compared to DNA footprinting, EMSA has:
A) Higher resolution
B) Lower resolution but easier method ✅
C) Same resolution
D) No application
EMSA results may be affected by:
A) Gel temperature
B) Salt concentration
C) Protein purity
D) All of the above ✅
Comparative & Conceptual
EMSA differs from footprinting because EMSA:
A) Identifies exact binding site
B) Detects mobility shift only ✅
C) Uses DNase I
D) Requires denaturing gel
EMSA is also known as:
A) Band shift assay ✅
B) Dot blot
C) Slot blot
D) Western blot
In EMSA, complexes must remain intact during:
A) DNA labeling
B) Electrophoresis ✅
C) Autoradiography
D) Staining
EMSA can be quantitative when:
A) No competitor is used
B) Protein concentration is varied systematically ✅
C) Only agarose gel is used
D) DNA is unlabeled
EMSA can detect binding of:
A) DNA only
B) RNA only
C) DNA or RNA with proteins ✅
D) Proteins only
Miscellaneous
The buffer in EMSA helps maintain:
A) DNA cleavage
B) Protein-DNA interaction stability ✅
C) DNA methylation
D) Gel polymerization
EMSA cannot distinguish between:
A) Free DNA and bound DNA
B) Specific and non-specific binding
C) Proteins of similar size without supershift ✅
D) DNA and RNA
Overloading protein in EMSA may cause:
A) Strong specific band
B) Smearing or non-specific binding ✅
C) Faster migration
D) DNA degradation
EMSA is often used before footprinting to:
A) Sequence DNA
B) Confirm binding interaction exists ✅
C) Digest DNA
D) Label protein
EMSA is best described as:
A) DNA sequencing technique
B) DNA–protein interaction detection method ✅
C) Protein purification technique
D) RNA isolation method.

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