Western Blot Sample Preparation Guide

Key Principles for Protein Sample Preparation

1. 1. Salt Concentration Optimization: Maintain appropriate ionic strength to maximize protein solubility and reproducibility
   2. Contaminant Removal: Effectively eliminate interfering molecules including nucleic acids, polysaccharides, and lipids
   3. Temperature Control: Perform all procedures at 4°C to prevent proteolytic degradation (include protease inhibitors)
   4. Sample Storage: Aliquot and store at -80°C; avoid repeated freeze-thaw cycles
   5. Concentration Methods: Use ultrafiltration or lyophilization for low-concentration samples

Recommended Protein Extraction Protocols

●RIPA-Based Total Protein Extraction (Suspension Cells)

1. 1. Harvest cells (1×10⁷) by centrifugation (2,000g × 5 min)
   2. Wash twice with ice-cold PBS (1 ml)
   3. Resuspend pellet in lysis buffer:
   4. RIPA: 300 μl
   5. PMSF: 3 μl (1:100)
   6. Protease inhibitor cocktail: 0.3 μl (1:1000)
   7. Incubate on ice for 30-40 min with vortexing every 10 min
   8. Centrifuge at 10,000g for 10 min (4°C)
   9. Collect supernatant as total protein extract

●Loading Buffer Direct Extraction

1. 1. Collect 1×10⁶ cells by centrifugation
   2. Wash once with PBS
   3. Resuspend in 50 μl 2× loading buffer
   4. Boil for 10-15 min (repeat 2-3 times until viscosity disappears)
   5. Centrifuge briefly before loading 5 μl (50 μg/μl) per lane

●Adherent Cell Protein Extraction

1. 1. Remove culture medium and wash cells 3× with cold PBS
   2. Add 400 μl lysis buffer (with 1:100 PMSF)
   3. Scrape cells and incubate on ice for 30 min
   4. Centrifuge at 12,000g for 5 min (4°C)
   5. Aliquot and store at -20°C

●Tissue Protein Extraction

1. 1. Mince tissue in homogenizer with 400 μl lysis buffer
   2. Homogenize with intermittent cooling
   3. Centrifuge at 12,000g for 5 min (4°C)
   4. Store supernatant at -20°C

●Plant Protein Extraction (SDS-PAGE)

1. 1. Grind 1g tissue in liquid nitrogen
   2. Add 3.5 ml extraction buffer and incubate 3-4 hr on ice
   3. Centrifuge at 8,000g for 40 min (4°C)
   4. Collect supernatant

Extraction Buffer Composition:

• • 1M Tris-HCl (pH 8): 300 ml
  • Glycerol: 75 ml
  • PVPP: 6 g

●TCA-Acetone Precipitation (2D Electrophoresis)

1. 1. Lyophilize tissue powder
   2. Precipitate with cold acetone (containing 0.07% β-mercaptoethanol)
   3. Centrifuge and dry pellet
   4. Solubilize in lysis buffer (7M urea, 2M thiourea, 4% CHAPS)

Technical Tips

• • For difficult tissues, consider mechanical disruption (sonication or bead beating)
  • Include phosphatase inhibitors for phosphoprotein studies
  • Determine protein concentration before storage
  • For long-term storage, consider adding 10% glycerol

This protocol provides reliable protein extraction for most western blot applications. For specialized needs, please consult our technical support team.