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.

To prepare the membrane, cut PVDF membranes into 3-millimeter by 3-millimeter pieces using surgical scissors that were cleaned immediately prior to use. Place the pieces of membrane on aluminum foil, and add 2 to 5 microliters of ethanol to each piece. Before membranes are completely dry, add 2 to 5 microliters of the protein eluant to each piece, and air-dry them until the membrane surface becomes matte.

Then, transfer membranes into 1.5-milliliter tubes, and store them at 4 degrees Celsius. If using immediately, add 2 to 30 microliters of ethanol, which will make them hydrophilic. Remove the ethanol with a pipette. But before the membranes dry, completely add 200 microliters of DTT-based reaction solution to each tube, and incubate them at 56 degrees Celsius for one hour.

After incubation, replace the reaction solution with 300 microliters of iodoacetamide solution, and incubate in the dark for 45 minutes. Then, wash the membranes twice with distilled water, and once with 2% acetonitrile by vortexing for at least 10 seconds.

Working with dithiothreitol and acetonitrile can be extremely hazardous. A mask, glasses, and gloves should always be worn when performing the on-membrane digestion procedure.

Prepare trypsin according to manuscript directions, and add 100 microliters of trypsin reaction solution to each membrane. Incubate overnight at 37 degrees Celsius for digestion.

The next day, transfer the reaction solution into a clean 1.5-milliliter tube, and add 100 microliters of washed solutions to the membrane. Incubate the membrane at 60 degrees Celsius for two hours.

Then, collect the washed solution and mix it with the reaction solution. Add another 100 microliters of washed solution to the membrane, and sonicate it for 10 minutes. Then, collect the washed solution, and mix it with the reaction solution. Cover the test tube with a piece of laboratory film, and make small holes in the film with a needle.

Dry the solution using a vacuum concentrator, and dissolve the residue in 10 microliters of 0.2% formic acid. Centrifuge the tube at 12,000 x g for three minutes, and transfer the supernatant into a sample tube.

Analyze the sample using a mass spectrometer linked to a nano-LC HPLC system.