On-Membrane Protein Digestion to Prepare Co-Immunoprecipitated Proteins for Interaction Studies (Scientific Article Protocol) | JoVE | Translated to Japanese (2024)

Encyclopedia of Experiments

Overview

This video demonstrates co-immunoprecipitated protein complexes on a PVDF membrane for protein-protein interaction analysis. The reduced proteins from complexes were treated with trypsin to cleave the individual proteins at the smaller peptides, then, the remaining peptides were extracted from the PVDF membrane. The pulled peptide derived from both proteins in the complex was then dried and resuspended in a low concentration of formic acid for further analysis.

Protocol

1. Immunoprecipitation

NOTE: We used non-sodium dodecyl sulfate (SDS) lysis buffer and citrate elution, as described in the following sections. However, the use of an alternative in-house immunoprecipitation technique may be also applicable for preparing LC-MS/MS samples.

  1. Transfect cultured cells with vectors encoding an epitope tag alone or a fusion protein. For acquiring representative data, transfer J774 cells (1 x 106) with vectors encoding a green fluorescent protein (GFP) alone or GFP-fused Capn6 (calpain-6 gene) using cationic liposomes.
  2. Conjugate antibody to magnetic beads.
    1. For this purpose, mix anti-GFP antibody (2 μL/reaction) with magnetic beads (25 μL/reaction) in 500 µL of citrate phosphate buffer (24.5 mM citric acid, 51.7 mM dibasic sodium phosphate; pH 5.0) in a 1.5 mL test tube.
    2. Rotate the mixture at 50 rpm for 1 h at room temperature. Then, wash the IgG-conjugated beads three times with citrate phosphate buffer containing 0.1% Polyoxyethylene (20) sorbitan monolaurate.
  3. Lyse the transfected cells using an appropriate lysis buffer. For acquiring representative data, lyse the transfected cells for 30 min on ice in 400 µL of lysis buffer (50 mM Tris-HCl; pH 7.5, 120 mM NaCl, 0.5% poly(oxyethelene) octylphenyl ether) containing 40 μmol/L phenylmethylsulfonyl fluoride, 50 μg/mL leupeptin, 50 μg/mL aprotinin, 200 μmol/L sodium orthovanadate, and 1 mM ethylene glycol tetraacetic acid (EGTA) in 1.5 mL test tubes 24 h after transfection.
  4. Clear the lysate by centrifuging at 15,000 xgfor 5 min and collect the supernatant.
  5. Preclear the lysate. Add unlabeled magnetic beads (25 μL/reaction) in a 1.5 mL-test tube. Wash the beads three times with 500 µL of citrate phosphate buffer. After the removal of the citrate phosphate buffer in the final wash, add the cell lysate over the magnetic beads. Rotate the mixture using a rotator at 50 rpm for 30 min at room temperature.
  6. Place the test tube on a magnetic stand for 5 min for magnetic separation, and collect the cell lysate. Use of the magnetic stand designated by the manufacturer is recommended.
  7. Bind the target proteins to the conjugated beads. Place the immunoglobulin (Ig)G-conjugated beads on a magnetic stand for 5 min for magnetic separation, discard the citrate buffer, and then add the cell lysate to the separated beads.
  8. Rotate the mixture at 50 rpm for 1 h at room temperature.
  9. Separate the target proteins from free non-target proteins. Wash the beads three times using 500 µL of citrate phosphate buffer containing 0.1% Polyoxyethylene (20) sorbitan monolaurate. After the final wash, add 30 µL of citrate buffer (pH 2–3), and incubate for 5 min at room temperature to elute the target proteins.
    NOTE: If recovery from the immunoprecipitation is insufficient, the use of alternative epitope tags, such as FLAG or HIS, may improve the efficiency. If the efficiency of the elution is insufficient, the use of other eluants, such as an SDS-based solution, may improve the efficiency.

2. On-membrane digestion of proteins

NOTE: Using protein-free materials and equipment is necessary to avoid contamination with exogenous proteins. In addition, it is recommended that the operator wear a surgical mask and gloves to avoid contamination by human proteins.

  1. Cut PVDF membranes into 3 mm x 3 mm pieces using surgical scissors that have been wiped with methanol and dried immediately prior to use.
  2. Add 2–5 µL of ethanol to the pieces of PVDF membrane on clean aluminum foil.
  3. Before they dry completely, add the eluant (2–5 µL each, 4–6 pieces per sample) onto the hydrophilic PVDF membranes, and then air-dry the membranes until the membrane surface becomes matte. Dried membranes can be stored at 4 °C.
  4. Transfer all membranes into 1.5 mL plastic tubes, add 20–30 µL of ethanol to make the membranes hydrophilic, and then remove the ethanol using a pipette.
  5. Before the membrane dries out completely, add 200 µL of dithiothreitol (DTT)-based reaction solution (80 mM NH4HCO3, 10 mM DTT, and 20% acetonitrile) and incubate it at 56 °C for 1 h.
  6. Replace the reaction solution with 300 µL of iodoacetamide solution (80 mM NH4HCO3, 55 mM iodoacetamide, and 20% acetonitrile), and incubate for 45 min at room temperature in the dark.
  7. Wash the membranes twice with 1 mL of distilled water and once with 1 mL of 2% acetonitrile by vortex mixing for >10 s.
  8. Dissolve lyophilized MS-grade trypsin (Table of Materials) directly in the reaction solution (30 mM NH4HCO3containing 70% acetonitrile). Add 100 µL of the reaction solution containing 1 μg of trypsin (Table of Materials) (30 mM NH4HCO3containing 70% acetonitrile) and incubate at 37 °C overnight.
  9. Transfer the reaction solution containing the tryptic digests into a clean 1.5 mL test tube using a pipette.
  10. Add 100 µL of wash solution (70% acetonitrile/1% trifluoroacetic acid) to the membrane and incubate it at 60 °C for 2 h. Collect the wash solution and mix it with the reaction solution. Add another 100 μL of wash solution to the membrane and sonicate it for 10 min. Then collect the wash solution and mix it with the reaction solution.
  11. Cover the test tube with a piece of laboratory film and make a couple of small holes with a needle. Dry the solution using a vacuum concentrator.
  12. Dissolve the residue in 10 µL of 0.2% formic acid. After centrifugation (12,000 ×g, 3 min at room temperature), transfer the supernatant into a sample tube.
    NOTE: The washes are the critical steps of this section. During on-membrane protein digestion, the washing of the membranes containing the immobilized proteins after reduction and alkylation with distilled water, followed by 2% acetonitrile, for more than 10 sec each, is critical for the removal of the reagents.

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Materials

NameCompanyCatalog NumberComments
AcetonitrileWako014-00386
Citric acidWako030-05525
DTTNacalai tesque14112-94
Dynabeads protein GThermo Fisher Scientific10003D
Formic acidWako066-00461
IodoacetamideWako095-02151
Lipofectamine 3000Thermo Fisher ScientificL3000008
Living Colors A.v. Monoclonal Antibody (JL-8)Clontech632380
NaClWako191-01665
NH4HCO3Wako018-21742
Nonidet P-40SigmaN6507Poly(oxyethelene) octylphenyl ether (n=9)
Protease inhibitor co*cktailSigmaP8465
Sequencing Grade Modified TrypsinPromegaV5111Trypsin
Sodium orthovanadateSigmaS6508
Sodium phosphate dibasic dihydrateSigma71643
TFAWako206-10731
TrisWako207-06275
Tween-20Wako160-21211

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On-Membrane Protein Digestion to Prepare Co-Immunoprecipitated Proteins for Interaction Studies (Scientific Article Protocol) | JoVE | Translated to Japanese (2024)
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