(PP)-InsP Affinity Probes for Target Characterization by Immunoblotting and Mass Spectrometry.

Pulldown experiments isolate molecular interactions using a "bait" molecule on solid supports, often leveraging the biotin-streptavidin system. Here, a streamlined workflow is described, which employs biotinylated inositol phosphate (InsPs) and inositol pyrophosphate (PP-InsP) probes to enrich target proteins from complex proteomes. The reagents are first immobilized onto streptavidin-coated beads, then exposed to cell lysates, and subsequently washed to remove nonspecific interactions. The enriched proteins are then eluted and analyzed via western blot or quantitative mass spectrometry. This approach leverages biotin-tagged probes to enhance coupling efficiency, simplify workflows, and enable diverse applications.