On-Membrane Protein Digestion to Prepare Co-Immunoprecipitated Proteins for Interaction Studies

To perform on-membrane digestion, take a solution of co-immunoprecipitated protein complexes, each containing a recombinant GFP-tagged target protein associated with its partner protein.

Transfer the proteins to a tube containing a pre-wet polyvinylidene difluoride, PVDF membrane, and incubate. The PVDF membrane's hydrophilic surface helps to transfer the proteins onto the membrane.

Transfer the protein-containing membrane to a fresh tube. Add a reducing agent-containing buffer to reduce the disulfide bonds between the cysteine residues, leading to denaturation of the individual proteins in the complexes without disturbing their interaction.

Replace this solution with an alkylating reagent, which alkylates the sulfhydryl group of both proteins in the complexes and prevents the reformation of disulfide bonds. Wash the membrane to remove any excess reagent.

Treat the membrane with a buffer containing trypsin — a proteolytic enzyme. Trypsin cleaves the GFP-tagged target protein and its partner protein at the arginine and lysine residues' carboxyl terminal, releasing the smaller peptides into the supernatant. Transfer the supernatant into a fresh tube.

Wash the membrane with an acidic solution, dissociating any bound peptides from the membrane, ensuring complete extraction. Pull the peptide-containing fractions into a single tube. Desiccate via vacuum drying.

Resuspend the residues in a low concentration of formic acid, dissolving the peptides, which are ready for protein-protein interaction identification.