Membrane and Cytoplasmic Protein Extraction Kit IC-7910

Product Introduction  

This kit enables the isolation and extraction of membrane proteins (including plasma membrane, mitochondrial membrane, endoplasmic reticulum membrane, etc.) and cytoplasmic proteins in approximately 90 minutes. Suitable for cultured cells (20-50 million) or tissues (~100 mg), it can also handle smaller sample sizes (e.g., 30-50 mg of tissue). Cells are lysed via homogenization or freeze-thaw cycles to avoid excessive membrane disruption while preserving protein integrity. A microscopy-based verification step ensures 70-80% cell lysis efficiency, allowing optimization of homogenization cycles. Extracted proteins are compatible with downstream applications such as SDS-PAGE, Western blotting, and phosphorylation assays. The membrane protein extraction reagent contains protease/phosphatase inhibitors (e.g., PMSF, EDTA), making it unsuitable for enzyme activity assays.  

Protocol (Summary)  

Pre-experiment Preparation  

1. Reagent Handling: Thaw Reagents A/B at room temperature, then immediately place on ice. Add PMSF (final concentration: 1 mM) before use.  

2. Sample Quantity Reference:  

   - Cells: 20-50 million (adherent/suspension cultures)  

   - Tissues: Recommended 100 mg (minimum 30-50 mg; reagent volumes remain unchanged).  

Sample Processing Steps  

A. Cell Samples  

1. Cell Harvesting  

1.1Adherent cells: Wash with PBS, then scrape or use EDTA-based detachment (avoid trypsin).  

1.2Suspension cells: Centrifuge to pellet.  

   Note: Remove all PBS residue after centrifugation (600g, 1 min).  

2. Cell Lysis: Add 1 mL Reagent A (with PMSF), incubate on ice for 10-15 min.  

B. Tissue Samples  

Mince into fine pieces, add 1 mL Reagent A (with PMSF), incubate on ice for 10-15 min.  

Cell Lysis Verification  

1. Homogenization (Recommended): Use a pre-chilled glass homogenizer (30-50 strokes on ice). Verify by microscopy: Examine 2-3 µL lysate for intact cells (visible nuclei or intact morphology). Target: 70-80% lysis. Record homogenizer model/strokes for reproducibility.  

2. Alternative: Freeze-Thaw: Perform 2 cycles (liquid nitrogen↔room temperature). Check lysis efficiency; repeat if needed.  

Fractional Centrifugation  

1. Remove Nuclei/Unlysed Cells: Centrifuge at 700g, 10 min (4¡æ). Carefully transfer supernatant (leave 30-50 µL to avoid pellet carryover).  

2. Cytoplasmic Protein Isolation: Centrifuge supernatant at 14,000g, 30 min ¡ú Collect supernatant (cytoplasmic fraction), store at −70¡æ.  

   Membrane Protein Isolation: Resuspend pellet in 200-300 µL Reagent B: Vortex 5 sec + ice incubation 5-10 min, repeat 1-2x ¡ú Centrifuge at 14,000g, 5 min ¡ú Supernatant contains membrane proteins, store at −70¡æ.  

Key Notes  

1. Yield Reference:  

   - Cytoplasmic protein: 5-30 mg per 50 million cells.  

   - Membrane protein: 0.3-3 mg per 50 million cells.  

2. Inhibitor Interference: Membrane protein extracts contain protease/phosphatase inhibitors and are unsuitable for enzyme activity assays but ideal for phosphorylation studies.  

3. Technical Tips:  

   - Retain a small supernatant layer during centrifugation to avoid cross-contamination.  

   - For higher membrane protein yield, gently resuspend pellet with minimal carryover.  

Troubleshooting  

- Low Yield: Increase homogenization strokes/freeze-thaw cycles; confirm >70% lysis.  

- Membrane Protein Contamination: Avoid pellet during 700g centrifugation; increase vortexing during membrane protein extraction.  

Storage  

- Kit: −20¡æ, stable for 1 year.  

- Protein Samples: −70¡æ for long-term storage.  

Safety  

Wear lab coats and gloves. For research use only.

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