How to Prepare RIPA Lysis Buffer
Strong denaturing lysis buffer for total protein extraction, good for Western blot
Components
| Chemical | Concentration |
|---|---|
| Tris-HCl | 50 mM (pH 7.4) |
| NaCl | 150 mM |
| NP-40 | 1% v/v |
| Sodium deoxycholate | 0.5% w/v |
| SDS | 0.1% w/v |
Preparation Strategy
Method: stock_then_dilute
Reason: Multiple components with masses <1g require stock solution approach for accurate measurement
Preparation Table
| Component | Conc. | Amount | Unit |
|---|---|---|---|
| Tris-HCl (pH 8.0) | 50 mM | 6.06 | g |
| NaCl | 150 mM | 8.77 | g |
| NP-40 (Igepal CA-630) | 1% (v/v) | 10 | mL |
| Sodium deoxycholate | 0.5% (w/v) | 5.0 | g |
| SDS | 0.1% (w/v) | 1.0 | g |
Procedure
- 1Add approximately 800 mL of ultrapure water to a 1 L beaker and begin gentle stirring
- 2Add 6.06 g Tris-HCl and stir until completely dissolved
- 3Add 8.77 g NaCl and stir until completely dissolved
- 4Add 5.0 g sodium deoxycholate slowly while stirring, allow extra time for complete dissolution
- 5Sprinkle 1.0 g SDS slowly onto the solution surface to minimize foaming, stir gently until dissolved
- 6Allow any foam to completely subside before proceeding
- 7Add 10 mL NP-40 and mix thoroughly by gentle inversion or slow stirring
- 8Verify pH is 8.0 ± 0.1 using pH meter, adjust if necessary with small amounts of HCl or NaOH
- 9Transfer to 1 L volumetric flask and bring to final volume with ultrapure water
- 10Mix thoroughly by inversion
pH Adjustment
Solution should be pH 8.0. If adjustment needed, use 1 M HCl to lower pH or 1 M NaOH to raise pH, adding dropwise with constant stirring. Allow 5-10 minutes equilibration between additions.
Sterilization
Filter through 0.22 μm filter into sterile containers. Do not autoclave as SDS will precipitate and detergents may degrade at high temperature. Use pre-sterilized filtration system and sterile storage bottles.
Safety
Level 2 safety required. Wear nitrile gloves, safety glasses, and lab coat. Use fume hood when handling SDS powder to avoid inhalation. NP-40 and deoxycholate are mild irritants. SDS is a moderate skin and eye irritant. Avoid skin contact with detergent solutions.
Storage
Store at 4°C in tightly sealed bottles. Shelf life 6 months. If white precipitate forms at cold temperature, warm to room temperature and mix until redissolved before use. Do not freeze as detergents may precipitate irreversibly.
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Customize this solutionRelated Solutions
NP-40 Lysis Buffer
Mild non-denaturing lysis buffer, preserves protein-protein interactions for co-IP
Triton X-100 Lysis Buffer
Non-denaturing lysis buffer with Triton X-100, suitable for co-IP and enzyme assays
SDS Lysis Buffer
Strong denaturing lysis buffer, fully denatures all proteins
Urea Lysis Buffer
Chaotropic lysis buffer for membrane proteins and inclusion bodies
IP Lysis Buffer (Immunoprecipitation)
Mild lysis buffer designed to preserve protein-protein interactions for IP/co-IP
CHAPS Lysis Buffer
Zwitterionic detergent buffer for membrane protein solubilization preserving native structure