Electrophoretic mobility shift assay

This is technique used widely for protein separation from a mixture and is very easy and less costly method. Slides cover all essential points about EMSA and it is quite interesting to know that how it detect and separate different proteins and their mobility shift assay.

1. Electrophoretic mobility shift Assay Group no. 15 16604 Fatima Tahir 16630 Iqra akbar 16638 Hajra Illayas

2. 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.

3. 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.

4. 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 electrophoretic separation of a protein-DNA or protein-RNA mixture on a polyacrylamide or agrose gel for a short period . The speed at which different molecules move through the gel is determined by their size, charge, and sometime by shape .

5. 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. How it Works?

6. Working of EMSA

7. 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.

8. Protocol or steps we follow in EMSA 1- Making labeled DNA: It has three ways: 1- 2-

9. 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.

10. 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.                                                                                                                                                                                                                      

11. 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.

12. Mu lt iple protein binding: Super shift:

13. Control Study understudy and result

14. 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. 

15. It is used to study conformational changes in DNA . For example a EMSA 2-D variant with electron microscopy is used for the characterization of conformation. Analysis of cellular extracts for the presence of certain DNA-binding proteins. Bound and free DNA are separated using EMSA. It proves useful for stoichiometric analysis. Number of protein that bind per DNA fragment I t 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. Applications

16. Advantages of EMSA

17. Drawbacks of EMSA:

18. Conclusion: