How to Prepare RIPA Lysis Buffer

Strong denaturing lysis buffer for total protein extraction, good for Western blot

Components

ChemicalConcentration
Tris-HCl50 mM (pH 7.4)
NaCl150 mM
NP-401% v/v
Sodium deoxycholate0.5% w/v
SDS0.1% w/v

Preparation Strategy

Method: stock_then_dilute

Reason: Multiple small-mass components (<1g each) require stock solutions for accurate measurement

Preparation Table

ComponentConc.AmountUnit
Tris-HCl (1M stock)50 mM final25.0mL
NaCl (5M stock)150 mM final15.0mL
NP-40 (10% stock)1% v/v final50.0mL
Sodium deoxycholate (10% stock)0.5% w/v final25.0mL
SDS (10% stock)0.1% w/v final5.0mL

Procedure

  1. 1STOCK PREPARATION PHASE:
  2. 2Prepare 1M Tris-HCl (pH 7.4): Dissolve 12.11g Tris-HCl in 80mL water, adjust pH to 7.4 with HCl if needed, bring to 100mL
  3. 3Prepare 5M NaCl: Dissolve 29.22g NaCl in water, bring to 100mL total volume
  4. 4Prepare 10% NP-40: Mix 10mL NP-40 with 90mL water (mix gently to avoid foam)
  5. 5Prepare 10% sodium deoxycholate: Dissolve 10g sodium deoxycholate in water, bring to 100mL
  6. 6Prepare 10% SDS: Slowly sprinkle 10g SDS onto 90mL water surface, stir gently until dissolved, bring to 100mL
  7. 7FINAL BUFFER PREPARATION:
  8. 8In a 500mL volumetric flask, add 300mL distilled water
  9. 9Add 25.0mL of 1M Tris-HCl stock solution
  10. 10Add 15.0mL of 5M NaCl stock solution
  11. 11Add 50.0mL of 10% NP-40 stock (add slowly to minimize foam)
  12. 12Add 25.0mL of 10% sodium deoxycholate stock
  13. 13Add 5.0mL of 10% SDS stock (add last, mix gently)
  14. 14Mix thoroughly but gently to avoid excessive foaming
  15. 15Bring final volume to exactly 500mL with distilled water
  16. 16Mix again and allow foam to subside before use

pH Adjustment

pH should be 7.4 from Tris-HCl stock. Verify with pH meter after mixing all components. Adjust with small amounts of 1M HCl or 1M NaOH if needed, but avoid over-adjustment as detergents can affect pH readings.

Sterilization

Filter through 0.22 μm filter unit. DO NOT AUTOCLAVE - detergents will precipitate and SDS concentration will be altered by heat treatment.

Safety

Level 2 safety: Wear nitrile gloves, lab coat, and safety glasses. Work in fume hood when handling SDS powder. NP-40 and deoxycholate are membrane-disrupting detergents - avoid skin contact. SDS powder is irritating to respiratory tract.

Storage

Store at 4°C in tightly sealed container. SDS may crystallize at cold temperatures - warm to room temperature and mix before use if crystals appear. Detergent activity may decrease over time. Good for 3 months when properly stored.

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Related 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