**Experimental Reagents and Procedures for Co-Immunoprecipitation**
**RIPA Buffer Composition:**
The RIPA buffer is designed to efficiently extract proteins while maintaining their structural integrity. The key components include:
- **Tris-HCl (50 mM, pH 7.4):** Acts as a buffering agent to prevent protein denaturation.
- **NaCl (150 mM):** Helps reduce non-specific protein aggregation.
- **NP-40 (1%):** A non-ionic detergent that aids in cell lysis and protein extraction.
- **Sodium Deoxycholate (0.25%):** An ionic detergent that enhances protein solubilization.
- **EDTA (1 mM):** A chelating agent that inhibits metal-dependent proteases.
- **Protease Inhibitors:**
- **PMSF (1 mM):** Prepared in isopropanol (200 mM stock), it should be added just before use due to its instability in aqueous solution.
- **Leupeptin (1 μg/ml), Aprotinin (1 μg/ml), Pepstatin (1 μg/ml):** These are prepared in water and stored at -20°C.
- **Phosphatase Inhibitors:**
- **Na₃VO₄ (1 mM), NaF (1 mM):** Added to prevent dephosphorylation during the experiment. Note: Do not add phosphatase inhibitors if conducting kinase activity assays.
**Preparation of Modified RIPA Working Solution (100 ml):**
1. Dissolve 790 mg Tris-Base in 75 ml deionized water, then add 900 mg NaCl and adjust pH to 7.4 with HCl.
2. Add 10 ml of 10% NP-40.
3. Add 2.5 ml of 10% sodium deoxycholate and mix until clear.
4. Add 1 ml of 100 mM EDTA and dilute to 100 ml with water. Store at 2–8°C.
5. On the day of use, add protease inhibitors (leupeptin, aprotinin, pepstatin) and phosphate inhibitors (Na₃VO₄, NaF). PMSF should be added last, as it degrades quickly in solution.
**Cell Lysis and Immunoprecipitation Protocol:**
1. Pre-chill PBS and RIPA buffer. Prepare a cell scraper wrapped in plastic and kept on ice.
2. Wash cells twice with pre-chilled PBS, then remove excess liquid.
3. Add pre-chilled RIPA buffer (1 ml per 10ⷠcells or 0.5 ml per 5×10ⶠcells).
4. Scrape cells into a 1.5 ml EP tube and incubate on ice for 5 minutes using a horizontal shaker.
5. Centrifuge at 14,000 g for 15 min at 4°C. Transfer the supernatant to a new tube.
6. Prepare Protein A agarose beads by washing them twice with PBS and resuspending in 50% PBS.
7. Incubate with beads for 10 min at 10°C to block non-specific binding. Centrifuge and discard beads.
8. Measure protein concentration using the Bradford assay. Dilute the lysate at least 1:10 to reduce detergent interference.
9. Add primary antibody (diluted according to cell line and target protein) and incubate overnight at 4°C or 2 hours at room temperature.
10. Add Protein A agarose beads and incubate for another 1 hour or overnight.
11. Wash beads three times with cold RIPA buffer, then resuspend in 2× loading buffer. Boil for 5 minutes and centrifuge.
12. Run samples on SDS-PAGE, stain with Coomassie blue, and analyze the target bands.
13. For further identification, excise the band, digest with trypsin, and perform LC-MS/MS analysis.
This detailed protocol ensures efficient and reproducible immunoprecipitation, allowing for accurate detection and characterization of protein interactions. Always check reagent stability and storage conditions to maintain experimental consistency.
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