Preparation of Cross-Linked Cellular Extracts with Formaldehyde

Abstract

Formaldehyde is a powerful cross-linking agent that elicits protein–protein and protein–nucleic acid cross-links. This protocol describes the formaldehyde cross-linking of intact cells followed by either the preparation of a whole-cell extract by sonication or the preparation of nuclear and cytoplasmic extracts by fractionation. Cross-linked extracts are treated with high salt or sodium dodecyl sulfate (SDS) to disrupt non-cross-linked aggregates. Protein–protein and protein–nucleic acid interactions can then be studied by purifying the components of interest from the extracts (e.g., using immunoprecipitation). Formaldehyde cross-links are reversible by heat.