Analysis of Direct Interaction between Viral DNA-binding Proteins by Protein Pull-down Co-immunoprecipitation Assay

Analysis of Direct Interaction between Viral DNA-binding Proteins by Protein Pull-down Co-immunoprecipitation Assay

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This protocol analyzes the direct interaction between two DNA-binding proteins by pull-down co-immunoprecipitation.One of the proteins is overexpressed in E.coli as HA-tagged recombinant protein and cell-free extracts Knife Rack are immunoprecipitated in HA-affinity resin.Cell extracts are treated with nuclease to degrade DNA and RNA, which rules out nucleic acid-mediated indirect interaction.Then, a second immunoprecipitation step is performed using the purified putative partner protein.

Co-immunoprecipitated proteins can be detected either by Coomassie Blue staining and/or Western blotting Art (WB) if a specific antibody is available.Moreover, many DNA/RNA binding proteins are highly electropositive, which can hinder WB under standard conditions, as has been shown in histones and histone-like proteins.In this case, we show that the high isoelectric point of the putative partner results in a poor transfer.Tips to troubleshot WB transfer of highly electropositive DNA-binding proteins are provided.