This plan only provides a guide, please modify it to meet your specific needs.
(1) DSP-crosslinking and Immunoprecipitation to Isolate Weak Protein Complex[1]:
A. Preparing cells expressing proteins of interests (POIs)
1. Plate 8.0×105 HeLa cells in 10cm dish per sample (10mL of DMEM containing 10% FBS per dish).
2. Incubate the cells at 37°C, 5% CO2 for 24h.
3. Transfect plasmids for FLAG-HA-tagged POI expression.
4. Incubate the cells at 37°C, 5% CO2 overnight.
B. Preparing Wash buffer and IP buffer
1. On the day of DSP-crosslinking and immunoprecipitation, prepare Wash buffer and IP buffer before the crosslinking. Keep them on ice.
C. DSP-crosslinking
1. Prepare 100mM DSP just prior to DSP-crosslinking.
2. Dilute 100mM DSP to 0.1mM DSP in pre-warmed (37°C) PBS.
3. Rinse the dishes twice with 5mL of pre-warmed (37°C) PBS/dish.
4. Discard the PBS from the dishes.
5. Add 5mL of 0.1mM DSP (prepared in step C2) in each dish.
6. Incubate the dishes at 37°C for 30min in a CO2 incubator.
D. Preparing beads for the first IP
1. During the DSP-crosslinking (step C6), prepare 40μL of Dynabeads Protein G/sample in one 1.5mL tube.
2. Wash the Dynabeads Protein G with the same amount of Wash buffer three times: Set the tube containing Dynabeads Protein G on a magnetic stand and let the beads accumulate onto the magnet. Discard the supernatant with a pipette. Add ice-cold Wash buffer. Remove the tube from the magnetic stand. Resuspend the beads by pipetting up and down. Repeat step D2 twice more.
3. Set the tube containing the washed beads on a magnetic stand and discard the supernatant.
4. Resuspend the beads with ice-cold IP buffer.
5. Add 1μL of anti-FLAG antibody per sample.
6. Incubate the beads on a rotating incubator at 4°C for 1h.
E. Stopping DSP-crosslinking
1. After the 30min incubation at step C6, discard the 0.1mM DSP and rinse the dishes once with 5mL of pre-warmed (37°C) PBS/dish.
2. Discard the PBS and add 5mL of STOP solution (room temperature)/dish.
3. Incubate the dishes at room temperature for 15min.
F. First IP
1. Discard the STOP solution from the dishes.
2. Rinse the dishes twice with 5mL of ice-cold PBS/dish.
3. Discard the PBS from the dishes.
4. Add 500μL of ice-cold IP buffer/dish.
5. Scrape off the cells with a scraper and transfer them with all the IP buffer on the dish into a 1.5mL tube.
6. Resuspend the cells by pipetting up and down.
7. Incubate the tubes on ice for 10min to complete cell lysis.
8. Centrifuge the tubes containing the lysates at 20,000×g at 4°C for 5min.
9. Transfer 500μL of the supernatant to a new 1.5mL tube for IP.
10. Add 40μL of anti-FLAG-antibody-bound beads (prepared at step D6) into the tube containing the supernatant.
11. Incubate the tubes on a rotating incubator at 4°C for 2h.
G. Preparing beads for the second IP
1. Approximately 30min before finishing the 2h incubation at step F11, prepare and wash new Dynabeads Protein G, as in steps D1 to D4.
2. Add 1μL of anti-HA antibody per sample.
3. Incubate the beads on a rotating incubator at 4°C for 1h.
H. Protein elution with FLAG peptides
1. Prepare FLAG-elution buffer by diluting FLAG peptide stock solution with TBS and keep it on ice.
2. After the 2h incubation at step F11, wash the beads with ice-cold 700μL of Wash buffer three times.
3. Discard the Wash buffer from the beads.
4. Resuspend the beads with 100μL of FLAG-elution buffer (prepared at step H1) gently by pipetting up and down.
5. Incubate the beads with FLAG-elution buffer on a rotating incubator at 4°C for 10min.
6. Set the tubes on a magnetic stand and transfer the supernatant (containing POI and POI-bound proteins) to new 1.5mL tubes.
7. Repeat steps H4 to H5 once more.
8. Set the tubes on a magnetic stand and transfer the supernatant to the tubes containing the first eluted proteins at step H6.
9. Add 300μL of ice-cold IP buffer to the collected supernatant.
I. Second IP
1. Mix the 500μL of supernatant at step H9 with 40μL of anti-HA-antibody-bound beads (prepared at step G3).
2. Incubate the beads on a rotating incubator at 4°C for 2h.
J. Final elution
1. After the 2h incubation at step I2, wash the beads with 700μL of ice-cold Wash buffer three times.
(2) DSP treatment of lysates of mouse brain[2]:
1. The lysates of mouse brain were treated with 5mM DSP for 30min to form intermolecular disulfide bonds, Pr-S-S-Pr in vitro and was stopped by addition of Tris buffer.
2. The lysates were then analyzed by the original biotin switch method and irreversible biotinylation procedures (IBP).
(3) Use DSP to prepare Au-TiO2 thin films for immunoassay experiments[3]:
1. Plasma-activated Au-TiO2 thin films were functionalized with DSP solutions, using various concentrations (0.4, 1, 4, and 10mg/ml) dissolved in DMSO solvent (dimethyl sulfoxide, anhydrous, ≥99.9%).
2. The immobilization time was 30min, followed by cleaning steps, including DMSO, PBS (Phosphate-Buffered Saline; pH 7.4; 10mM), and ethanol 96% solutions to remove any unbounded cross-linker molecules.
References:
[1] Akaki K, Mino T, Takeuchi O. DSP-crosslinking and Immunoprecipitation to Isolate Weak Protein Complex. Bio Protoc. 2022;12(15):e4478.
[2] Huang B, Chen C. Detection of protein S-nitrosation using irreversible biotinylation procedures (IBP). Free Radic Biol Med. 2010;49(3):447-456.
[3] Meira DI, Barbosac AI, Proença M, et al. Immobilizing antibody biorecognition layers on Au–TiO2 thin films: direct (physisorption) vs. DSP-crosslinking (chemisorption) surface functionalization. Journal of Physics D: Applied Physics. 2024;415401.
