NSC15520

Electrophoretic mobility shift assay detects protein-nucleic acid binding by incubating a labeled DNA or RNA probe with purified protein or cell extract, then separating free probe from slower-migrating protein-probe complexes on a native gel. For cancer cells, primary neurons, mouse tumor samples, intestinal organoids, inflammatory macrophages, or drug-treated samples, EMSA can measure transcription-factor DNA binding or RNA-binding protein activity in extracts, but it does not directly measure transcription, protein expression, or chromatin occupancy in intact cells. Specificity is judged by competition with unlabeled wild-type probe, failure of mutated or unrelated competitors to compete, and antibody supershift or disruption when the binding protein identity must be confirmed.

PCNA immunodetection measures proliferative activity by detecting proliferating cell nuclear antigen, a nuclear protein associated with DNA polymerase δ function and DNA replication. The assay readout is the proportion of PCNA-positive nuclei among total counted cells, but PCNA labeling is not identical to BrdU labeling because PCNA can mark late G1/early S-associated replication competence and may persist beyond active DNA synthesis depending on fixation and extraction conditions.

The bacterial reverse mutation assay detects point mutations that restore amino-acid prototrophy in auxotrophic Salmonella typhimurium or Escherichia coli tester strains; after exposure to a test article, mutagenic activity is read out as an increased number of revertant colonies on minimal agar compared with the vehicle control. The assay uses tester strains with different mutation targets so that base-substitution and frameshift mutagens can be detected, and testing is performed with and without exogenous mammalian metabolic activation because some chemicals require biotransformation to become mutagenic.