Skip to content
Login Register

#DNA-protein interactions

2 public questions tagged with this topic.

Transcript analysis

In ChIP, which is precipitated?

Chromatin immunoprecipitation investigates in vivo association of proteins with genomic DNA. Living cells are treated with formaldehyde, which cross-links proteins to DNA and proteins to proteins preserving physiological interactions. Chromatin is sheared by sonication or enzymatic digestion into 200-500 base fragments. An antibody specific to transcription factor, histone modification, or cofactor immunoprecipitates cross-linked chromatin, enriching bound DNA fragments. After reversal of cross-links and protease digestion, recovered DNA is analyzed. Thus final immunoprecipitate comprises protein-DNA complex, not naked DNA or RNA alone, reflecting chromatin context and occupancy.

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.

View Details
Transcript analysis

EMSA detects:

Electrophoretic Mobility Shift Assay is a classic in vitro technique for studying nucleic acid-protein interactions. A radiolabeled or fluorescent double-stranded DNA fragment containing putative protein binding motif is incubated with nuclear extract or purified protein. If specific binding protein recognizes sequence, formation of protein-DNA complex increases molecular mass and reduces net negative charge, causing retardation during non-denaturing polyacrylamide gel electrophoresis compared to free probe. The assay can also detect protein-protein interactions via supershift when additional partner increases complex size. It does not measure protein degradation or folding status directly.

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.

View Details