**Experimental Reagents and Procedures**
**1. RIPA Buffer Composition:**
RIPA buffer is a commonly used lysis buffer for protein extraction, containing several key components:
- **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 membrane disruption and protein solubilization.
- **Sodium deoxycholate (0.25%)**: An ionic detergent that enhances protein extraction; it should be prepared in water and stored at 2–8°C.
- **EDTA (1 mM)**: A calcium chelator that inhibits metalloproteases and phosphatases.
- **PMSF (1 mM)**: A protease inhibitor, prepared in isopropanol (200 mM stock), and added just before use due to its instability in aqueous solution.
- **Leupeptin (1 μg/ml), Aprotinin (1 μg/ml), Pepsin Inhibitor (1 μg/ml)**: Protease inhibitors, stored in water at -20°C.
- **Na₃VO₄ (1 mM)** and **NaF (1 mM)**: Phosphatase inhibitors, used to block phosphatase activity during kinase or phosphatase assays.
**Note:** Avoid adding sodium cholate in kinase experiments, as ionic detergents can interfere with enzyme activity. Also, when performing phosphatase assays, do not include phosphate inhibitors like Na₃VO₄ and NaF.
**2. Preparation of Modified RIPA Working Solution (100 ml):**
- Dissolve 790 mg Tris base in 75 ml deionized water, then add 900 mg NaCl and adjust the pH to 7.4 using HCl.
- Add 10 ml of 10% NP-40, followed by 2.5 ml of 10% sodium deoxycholate. Stir until clear.
- Add 1 ml of 100 mM EDTA, then dilute to 100 ml with deionized water. Store at 2–8°C.
- On the day of use, add protease and phosphatase inhibitors:
- Leupeptin, Aprotinin, Pepsin Inhibitor: 100 μl each (final concentration ~1 μg/ml)
- PMSF: 500 μl (final concentration ~1 mM)
- Na₃VO₄ and NaF: 500 μl each (final concentration ~1 mM)
**3. Cell Lysis and Immunoprecipitation Protocol:**
- Pre-chill PBS and RIPA buffer, and prepare a cell scraper wrapped in plastic and kept on ice.
- Wash cells twice with pre-chilled PBS, then scrape them off the dish into pre-chilled RIPA buffer (1 ml per 10â· cells).
- Incubate at 15°C for 5 minutes on a horizontal shaker. Centrifuge at 14,000 × g for 15 min at 4°C. Transfer the supernatant to a new tube.
- Prepare Protein A agarose beads by washing them twice with PBS and resuspending at 50% in PBS.
- Incubate the lysate with 100 μl of 50% agarose beads for 10 min at 10°C to remove non-specific binding. Centrifuge again and discard the beads.
- Quantify the protein concentration using the Bradford assay. Dilute the sample at least 1:10 to reduce detergent interference.
- Incubate the diluted lysate with the appropriate primary antibody (e.g., rabbit anti-protein of interest) at 4°C overnight or at room temperature for 2 hours.
- Add 100 μl of Protein A agarose beads and incubate for another 1–2 hours.
- Wash the beads three times with pre-chilled RIPA buffer.
- Resuspend the beads in 2× loading buffer, boil for 5 minutes, and centrifuge to collect the supernatant for SDS-PAGE analysis.
**4. Co-Immunoprecipitation and Western Blotting:**
- Wash cells in phosphate-buffered saline, scrape into EBC lysis buffer, and centrifuge at 4°C.
- Add 30 μg of specific antibody and incubate at 4°C for 1 hour.
- Add protein A-Sepharose beads and incubate for 30 minutes. Wash five times with NETN buffer.
- Boil the beads in 1× SDS loading buffer, load onto a gradient SDS-PAGE gel, and run overnight at 10 mA.
- Stain with Coomassie blue, excise the target band, and perform trypsin digestion.
- Analyze the resulting peptides using high-performance liquid chromatography (HPLC) and sequence them via automated Edman degradation on an ABI 477A or 494A sequencer.
This detailed protocol ensures efficient protein extraction, immunoprecipitation, and identification of interacting proteins.
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