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#protein-DNA complexes

2 public questions tagged with this topic.

DNA-Protein Interaction analysis

Which method allows immunoprecipitation of protein-DNA complexes?

Chromatin Immunoprecipitation is definitive technique for immunoprecipitating protein-DNA complexes from living cells. Cells are crosslinked with formaldehyde to covalently stabilize transient protein-DNA contacts. Chromatin is sheared by sonication or micrococcal nuclease to fragments of 200-500 base pairs, incubated with antibody specific to transcription factor or histone modification, captured on protein A or G beads. After extensive washing, crosslinks are reversed, proteins digested, DNA purified for PCR or sequencing. This in vivo approach directly identifies genomic loci occupied, unlike in vitro EMSA or Western blot.

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 slower migrating band in EMSA suggests:

In EMSA, electrophoretic mobility under non-denaturing conditions reflects size and charge. Free double-stranded DNA probe migrates quickly toward anode owing to phosphate backbone negativity. When sequence-specific DNA-binding protein recognizes probe, resulting complex possesses substantially higher molecular weight, altered conformation and lower charge density, causing markedly slower migration. Consequently, complex appears as higher, slower band compared to free probe. This retarded band confirms formation of DNA-protein complex, useful for transcription factor analysis. DNA degradation would create fast smear, contamination alone would not yield discrete specific shift.

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