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#electrophoretic mobility shift assay

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DNA-Protein Interaction analysis

EMSA is also known as:

Electrophoretic Mobility Shift Assay, abbreviated EMSA, is formally known as electrophoretic mobility shift assay, also called gel mobility shift or gel retardation assay. Name directly describes underlying principle: association of protein with labeled DNA probe retards electrophoretic mobility in non-denaturing polyacrylamide gel, yielding shifted band. It is fundamental technique for demonstrating transcription factor binding, measuring affinity and assessing specificity with competitors. Reporter assay measures promoter activity via reporter gene, trap assay implies different purpose. Correct expansion is essential terminology for competitive examinations in molecular biology.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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DNA-Protein Interaction analysis

A supershift in EMSA indicates:

Supershift assay extends EMSA to identify protein composition within DNA-protein complexes. Labeled DNA probe is first incubated with nuclear extract to form specific retarded complex. Then antibody recognizing candidate transcription factor is added. If antigen is present within complex, antibody binds, dramatically increasing molecular weight, bulk and hydrodynamic drag, producing even slower migrating band above original shift. Appearance of supershifted species confirms presence of specific protein and indicates multiprotein assembly or protein-protein interaction on DNA. Free probe migrates normally, degraded probe shows loss rather than additional retardation.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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