Electrophoretic Mobility Shift Assay (EMSA)

October 06, 2015

Texas Tech University Health Science Center
Internal Medicine
Postdoctoral Researcher

Overall

Quality of Results

Ease-of-Optimization

What do these ratings mean?
Write a Review

Company:

Thermo Scientific

Product Name:

LightShift Chemiluminescent EMSA kit

Catalog Number:

20148

EMSA is performed to study the protein-DNA interactions which exist as a complex. Due to the protein-DNA complex, it will migrate slower than the non-bound DNA in a native gel which results in a shift due to the labelled DNA probe.

Experimental Design and Results Summary

Application

EMSA by using native polyacrylamide gel

Starting Material

Mouse cell lysate

Protocol Overview

It contains 7 steps: 1)Planning of the binding reactions 2)Preparation of native polyacrylamide gel 3) Preparation of binding reactions 4) Perform electrophoresis 5) Transfer of binding reactions to the nylon membrane 6) UV crosslink the transferred DNA to membrane 7) Detection ob Biotin-labelled DNA by chemiluminescence

Tips

After transferred onto the membrane, UV cross link should be given as early as possible.

Results Summary

This is a nonisotopic way of detecting the DNA-protein interactions. The probe/DNA containing the binding site of our inetrest was labelled with biotin and allowed to incubate with the nuclear extract then transferred onto the nylon membrane and detected using the Strepavidin-Horseradish Peroxidase conjugate and chemiluminescence substrate.

Additional Notes

None

Related Categories

Image Gallery

Summary

The Good

It's very easy to optimize the technique and the results are very reliable.

The Bad

The chemiluminescence substrate is very strong but not strong enough. The signal is for a very short amount of time so we have to detect as early as possible.

The Bottom Line

This EMSA kit is very good and detects the protein-DNA interactions by avoiding use of the radioactive isotope. Basically it is a one day experiment if you are ready with all the materials required. It is highly recommended.

Join the discussion