Proteins nonspecifically precipitated by anti-TREK1 beads and identified by mass spectrometry
9.3. Method
9.3.1. Preparation of Affinity-Purified Anti-TREK1 Antibodies
3. Pre-absorb antibodies directed against the GST-tag by incu-bating 10 mL of serum with GST aminolink beads (1 mL at 5 mg/mL) during 2 h at 22°C. After centrifugation, collect the supernatant and incubate with GST-TREK1 aminolink beads (1 mL at 1 mg/mL) for 1 h at 22°C.
4. Wash the GST-TREK1 aminolink beads with 5-column volumes.
5. Elute the affinity-purified anti-TREK1 antibodies using 2 mL of glycine buffer.
6. Neutralize the pH of the solution containing the eluted anti-bodies using NaOH.
7. Determine the concentration of anti-TREK1 antibodies using a Bradford assay (Bio-Rad) (see Note 2).
1. Gently spin down 0.3 mL of protein A-sepharose beads (50/50 slurry) with a mini desktop centrifuge (1,000×g, 2 min) and wash twice with PBS.
2. Re-suspend the beads in 0.3 mL of PBS and add 0.3 mg of puri-fied antibodies (here anti-TREK1 antibodies). Mix on a rocking table for 1 h at 22°C. Spin down (1,000g, 2 min) and save the supernatant for a Bradford assay to verify that all the antibodies have been adsorbed on the protein A-sepharose beads.
3. Wash the beads with 10 volumes of 0.2-M triethanolamine (twice) then spin down.
4. Re-suspend the beads in 20 volumes of freshly made 50-mM DMP in 0.2-M triethanolamine, pH 8.2 for covalent crosslinking of antibodies to protein A.
5. Gently rotate 45 min at 22°C, then spin down and suck off the liquid fraction.
6. For neutralization, re-suspend the beads in an equal volume of 50-mM ethanolamine, pH 8.2 and gently agitate on a rocking table 5 min at 22°C. Spin down and suck off the supernatant.
7. Re-suspend the beads in an equal volume of 50-mM eth-anolamine pH 8.2 and keep for 1 h with gentle agitation at room temperature.
8. Spin down, exchange ethanolamine for PBS (twice) and remove the supernatant.
9. Wash twice with 10 volumes of elution buffer (pH 11.6) to eliminate the antibodies that are not covalently crosslinked. Immediately after, wash in PBS. The beads are ready to use.
10. Antichannel beads can be stored at 4°C for several weeks/
months by adding sodium azide (final concentration: 0.05%) (see Note 3).
9.3.2. Preparation of the Anti-TREK1 Affinity Column
1. Sacrifice five WT and five TREK1−/− mice by decapitation and rapidly collect the whole brains by dissection.
2. Drop the brains into ice-cold homogenization solution (around 6 mL for five brains, total volume of 10 mL).
3. Homogenize with a tissue grinder (Potter Elvehjem, total clearance 0.1 mm), 12 up-and-down strokes at 1,000 rpm.
4. Dilute with 28 mL of 2-M sucrose solution, then add 12 mL of CaCl2 solution. Mix. The final concentration of sucrose is 1.25 M. Transfer 25 mL into two SW28 (25 × 89 mm) cen-trifuge tubes (Beckman).
5. On this 25-mL layer carefully pour a 10-mL layer of 1-M sucrose solution, then a last layer of 5 mL of 0.32-M sucrose solution above to form a three-layer discontinuous gradient.
6. Spin for 3 h at 100,000×g and 4°C (Beckmann ultracentrifuge).
7. The first white upper band is mainly formed by myelin. The pinkish band at the interface between 1.25- and 2-M sucrose layers contains synaptosomes, while the red pellet contains mitochondria, blood cells, nuclei and cell debris.
8. The myelin layer was carefully discarded and then the synap-tosomes were gently collected by pipetting.
9. The synaptosomes were diluted and gently homogenized in 30 mL of PBS and spun down 1for 5 min at 10,000×g to remove sucrose.
10. Finally, the synaptosomal pellets were re-suspended in 4 mL of PBS and used immediately or kept at −80°C after protein quantification using a Bradford assay (Bio-Rad).
1. Spin down 10 mg of synaptosomal proteins for 15 min at 10,000×g. Re-suspend the pellets in solubilization buffer (1 mL), gently agitate for 4 h at 4°C on a rocking table, then centrifuge for 30 min at 20,000×g at 4°C. Collect the supernatant contain-ing the solubilized membrane proteins and keep at 4°C.
2. Wash 50 µL of the beads twice with 500 µL of PBS containing 0.1% Triton-100.
3. Load the solubilized proteins (1 mL of supernatant) onto the beads (50 µL corresponding to 100 µg of antichannel IgG) and incubate overnight at 4°C with gentle agitation (see Note 4).
4. Load the beads and supernatant into a SigmaPrep spin column connected to a vacuum apparatus for aspiration of the superna-tant and wash buffer (Vac-Man laboratory vacuum manifold, Promega) (see Note 5).
5. Quickly wash the beads with 5 mL of ice-cold lysis buffer.
6. Remove the SigmaPrep spin column from the Vac-Man and place in an Eppendorf tube.
9.3.3. Synaptosomal Protein Preparation
9.3.4. Immunoprecipi-tation
7. Elute the bound proteins by adding 25 µL of Laemmli sample buffer and incubating for 10 min at 22°C with gentle agitation (see Note 6).
8. Spin down (1,000×g, 5 min) and keep the eluate for gel sepa-ration.
1. Load the precipitated proteins onto a commercial denaturing gel and separate for 1 h at 150–200 V.
2. Stain the gel using coomassie blue (see Note 7).
3. Cut each lane into ten fragments and identify the precipitated proteins via direct nanoLC-ESI-MS/MS analysis of trypsin-digested gel fragments (see Note 8).
1. The cytosolic carboxy-terminal (C-ter) domain of TREK1, immediately following the fourth transmembrane segment (M4), plays a key structural role in channel activation sug-gesting that this region may be involved in protein-protein interaction and regulation. To prevent an eventual competi-tion between the binding of the precipitating antibodies and the protein partners, we targeted the anti-TREK1 antibodies against the amino-terminal part of the channel.
2. Usually around 500 µg of specific antibodies are recovered from 10 mL of crude serum.
3. To check the quality of the affinity beads, different amounts of GST, GST-TREK1 and BSA proteins were incubated with 5 µL of beads. Precipitated proteins were analyzed by SDS-PAGE. The column was able to precipitate GST-TREK1 and to a minor extent, GST alone. The binding capacity was estimated to be close to 500 ng of GST-TREK1 per µL of beads.
4. By Western blot analysis, and using GST-TREK1 for compari-son and quantification, we estimated that 10 ng of solubilizable TREK1 channel was present in 1 mg of the total synaptosomal proteins. 10 mg of synaptosomal proteins corresponds to 100 ng of solubilized TREK1 channel.
5. At this step it is important to avoid allowing the column to dry out.
6. The Laemmli sample buffer MUST NOT contain any reduc-ing agent such as dithiothreitol (DTT) or β-mercaptoethanol (β-ME) to avoid reduction of interchain disulfide bridges and release of IgG chains in the eluate.
9.3.5. SDS-PAGE and Protein Identification