Researching protein-protein interaction is indispensable for understanding physiological processes in several field. In this video we introduce on-membrane digestion technique which allows the researchers to perform a comprehensive analysis of binding proteins by MS in a convenient manner. The main advantage of this technique is that we can improve the throughput of proteomic analysis for crude immunoprecipitants because separation of proteins by polyacrylamide gel electrophoresis is not necessary.
Begin conjugating the antibody to the magnetic beads, by mixing the anti-GFP antibody with the beads in 500 microliters of citrate phosphate buffer. Rotate the mixture at 50 RPM for one hour at room temperature. And then wash the conjugated beads three times with citrate phosphate buffer containing 1%polysorbate 20.
Lyse transfected cells according to manuscript directions. Scrape the dish and transfer the lysate into the 1.5 milliliter test tube. Then clear the lysate by centrifuging at 15, 000 x g for five minutes and collect the supernatant.
Prepare unlabeled magnetic beads by washing them three times with 500 microliters of citrate phosphate buffer. After the final wash, remove the buffer and add cell lysate to the beads. Rotate the mixture at 50 RPM for 30 minutes at room temperature.
Perform magnetic separation on the magnetic stand and collect the cell lysate. To bind the target proteins to the immunoglobulin conjugated beads, place the beads on the magnetic stand for five minutes. Discard the citrate buffer and add the cell lysate to the beads.
Rotate the mixture for one hour at 50 RPM. To separate target proteins from the free non-target proteins, wash the beads three times with 500 microliters of citrate phosphate buffer containing 1%polysorbate 20. After the final wash, add 30 microliters of citrate buffer, and let the beads sit for five minutes at room temperature to elute the target proteins.
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 two to five microliters of ethanol to each piece. Before membranes are completely dry, add two to five microliters of the protein eluent 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 four degrees Celsius. If using immediately, add two 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 ten seconds. Working with diaphanous-related 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 wash solutions to the membrane. Incubate the membrane at 60 degrees Celsius for two hours, then collect the wash solution and mix it with the reaction solution. Add another 100 microliters of wash solution to the membrane and sonicate it for 10 minutes.
Then collect the wash 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 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. Using this protocol, 17 proteins were identified in the Calpain-6 associated immunoprecipitant, 15 in the GFP-associated immunoprecipitant, and 11 were identified in both.
It's important to note that detection of at least one high fidelity peptide fragment is required for positive identification of a candidate protein. Proteins that precipitated in the GFP-expressing lysate as well as histones, exogenous and ribosomal proteins were omitted from the list. Washing the membranes before tryptic digestion with distilled water and 2%acetonitrile is the most important step in the procedure of on-membrane digestion over proteins.
Following this method, it is possible to confirm co-precipitation of target protein by conducting other experiments, such as immunoprecipitation and Western blot analysis. This simple method allows the researchers to explore the novel protein-protein interactions with convenient and high-sensitive analysis.
