Introduction to Immunoprecipitation Mass Spectrometry (IP-MS)
Data on Protein-Protein Interactions (PPIs) and Post-Translational Modifications (PTMs) proteomics significantly elevate the caliber of fundamental research articles. Immunoprecipitation Mass Spectrometry (IP-MS), a core tool in proteomic studies, is pivotal for screening and discovering novel interacting proteins or new post-translational modifications of target proteins within cells.
In a single IP-MS experiment, high-throughput identification and screening of multiple proteins that interact with the target protein can be achieved. This process unveils the components of protein complexes related to the studied target, establishing a unique protein-protein interaction database. This personalized database allows for more in-depth exploration of biological functions and molecular mechanisms, enhancing the depth of research in this field.
Experimental Procedure
1. Cell Lysis: Lyse cells using Lysis Buffer, optionally with sonication or by placing on ice for 30 minutes, aspirating cells every 10 minutes until visibly large granular cells are no longer observed.
Recommended amounts: 500 μL Lysis Buffer for cells in a dish smaller than 10 cm, 1 mL for a 10 cm dish, and 2 mL for a 15 cm dish (2e7 cells).
2. Centrifugation: Centrifuge in soft mode at 4 ℃, 20,000 ×g for 15 minutes.
3. Antibody Addition: Transfer the supernatant to a new 1.5 mL tube and add an appropriate amount of antibody.
Recommended amounts: 1 μg antibody for less than a 10 cm dish, 2 μg for a 10 cm dish, and 5 μg for a 15 cm dish.
4. Incubation: Invert and mix the EP tube at 4 ℃ for at least 2 hours.
5. Centrifugation (Round 1): Centrifuge in soft mode at 4 ℃, 20,000 ×g for 15 minutes.
6. Magnetic Bead Balancing: During the 5th centrifugation, balance the magnetic beads with Lysis Buffer three times.
7. Magnetic Bead Incubation: Transfer the supernatant from step 5 to a new tube and add an appropriate amount of magnetic beads.
Recommended amounts: 5 μL solid beads for less than a 10 cm dish, 5 μL for a 10 cm dish, and 15 μL for a 15 cm dish.
8. Extended Incubation: Invert and mix the EP tube at 4 ℃ for at least 2 hours or overnight.
9. Bead Separation: On a magnetic rack, let beads settle for 1 minute, remove the supernatant, or centrifuge at 1000 ×g for 2 minutes to separate agarose beads, removing the supernatant.
10. Washing: Add 1 mL Wash Buffer, invert and mix, then separate beads on a magnetic rack for 1 minute, or centrifuge at 1000 ×g for 2 minutes to remove the supernatant. Repeat washing at least three times.
11. Final Wash: Wash beads three times with Replacement Buffer (150 mM NaCl, 20 mM Tris-HCl, pH 7.5) or PBS, and mix 1/4 volume of bead suspension with a new EP tube for WB and SDS-PAGE verification.
12. Storage or Mass Spectrometry: Preserve the remaining 3/4 bead mixture without the supernatant; store at -20 ℃ or send for mass spectrometry.
13. SDS-PAGE Verification: Discard the supernatant from 1/4 bead mixture, add 50 μL Loading Buffer, boil for 10 minutes, and collect the upper solution after centrifugation.
14. Gel Electrophoresis: Run two SDS-PAGE gels with 20 μL samples each for WB and Coomassie/Silver staining.
15. Verification: WB reveals targeted protein bands, while Coomassie/Silver staining shows abundant bands in both experimental and control groups, indicating the normalcy of the IP experiment process.
Note
Buffer Composition:
The components of Lysis Buffer and Wash Buffer may vary slightly between laboratories. A general reference is provided below:
| 10x Lysis Buffer (1 L): | |
| 200 mM Tris-HCl pH 7.5 | 24.23 g Tris base, adjust to pH 7.5 |
| 1.5 M NaCl | 87.75 g powder |
| 10 mM EDTA | 20 mL 0.5M EDTA |
| 10%Triton X-100 (or NP-40) | 100 mL Triton X-100 (or NP-40) |
Western Blot (WB):
WB is a targeted detection method with multi-level antibody amplification, theoretically more sensitive than non-targeted detection by mass spectrometry. Therefore, for IP experiments requiring mass spectrometry, it is advisable to increase cell quantity. High-quality IP-MS studies typically use an initial cell quantity of 1e8 (approximately 10 cm dish x 10 or 15 cm dish x 5). We recommend a starting cell quantity for wild-type IP not less than 2e7/sample. For overexpressed tagged target proteins or endogenous target proteins with high expression levels, cell quantity can be reduced accordingly, but not recommended to be less than 1e7/sample.
IP-MS Experimental Design:
IP-MS recommends using a quantitative approach for screening interacting proteins. Therefore, consistency in cell and reagent quantities between control and experimental groups is crucial.
Antibody Validation:
Despite the availability of antibodies labeled for IP on the market, only a small portion proves effective. To save time and resources, it is recommended to conduct quality control at key experimental points. We suggest that after the initial IP experiment of the target protein, essential steps include WB and Coomassie/Silver staining for quality control.
