Your Step-by-Step Guide to Electrophoretic Mobility Shift Assay

1. Your Step-by-Step Guide to Electrophoretic
Mobility Shift Assay
—by Creative BioMart

2. • The Electrophoretic Mobility Shift Assay (EMSA), or
gel shift assay is a basic and rapid method to detect
protein complex with nucleic acids.

3. • 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.

4. • It is likewise connected to qualify an quantify
proteins that particularly tie to given nucleotides,
empowering to accommodate a wide range of
binding conditions.

5. Principles
• 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.

6. • DNA-complexes or RNA-complexes move slower than
non-bound probes. Isotope-labeled probes differ
depending on the binding protein studied, double-
stranded or single-stranded can both be ok. When
detecting DNA binding proteins such as
transcriptional regulatory factors, purified proteins,
partially purified proteins, or nuclear cell extracts can
be used.

7. • In the detection of RNA binding protein, according to
the location of the target RNA binding protein,
purified or partially purified protein, as well as
nuclear or cytoplasmic cell extract can also be used .

8. • The DNA or RNA fragments and oligonucleotide
fragments (specific) containing protein binding
sequences, and other non-relevant fragments (non-
specific), were used in competitive experiments to
determine the specificity of DNA or RNA-binding
proteins. Specific binding is determined according to
the characteristics and intensity of the complex in
the presence of competing specific and non-specific
fragments.

9. Experimental Materials
DNA samples
Reagents, kits
• [γ-32P]ATP, T4 polynucleotide
kinase, Nuclease-Free Water, T4
polynucleotide kinase buffer,
Ammonium acetate, TE, Anhydrous
ethanol, TBE buffer, steaming
water, Methylene bisacrylamide,
Acrylamide, glycerin, Ammonium
persulfate, TEMED
(tetramethylethylenediamine),
EMSA Gel-Shift binds buffer, BPB
Equipment
• Water bath, PCR,
Centrifuge, Electrophoresis,
Electrophoresis tank

10. Operating Method
• (1)Probe labeling
• (2)Probe Isolation
• (3)EMSA glue preparation
• (4) EMSA
• (5) Electrophoretic analysis

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