Western Blot/Tissue Preparation

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Contents

Overview

This is a protocol for preparing whole cell lysate from tissue, for western blot analysis.

Materials

RIPA Buffer

per 100ml: (final concentration)

  • 5ml of 1M Hepes, pH 7.6 (50 mM)
  • 200µL of 0.5M EDTA (1 mM)
  • 7 ml of 10% DOC (Na deoxycholate) (0.7%)
  • 10 ml of 10% NP-40 (IPGEL) (1%)
  • 10 ml of 5M LiCl or 2.12g powder (0.5 M)

Protease inhibitors

  • For example, Complete tabs from Roche [1]
  • Or [2]


Procedure

  1. Weigh tissue.
  2. Dice into very small pieces using a clean razor blade.
  3. Add 3 ml of ice cold RIPA buffer (supplemented with protease inhibitors) per gram of tissue.
  4. Further disrupt and homogenize tissue with a dounce homogenizer or a sonicator. Maintain temperature at 4° C throughout all procedures.
  5. Incubate on ice for 30 minutes.
  6. Transfer to microcentrifuge tubes, centrifuge at 10,000xg for 10 minutes at 4° C.
  7. Remove supernatant and centrifuge again. The super-natant fluid is the total cell lysate.

Notes

  • If necessary, use a longer centrifugation to obtain a clear lysate.

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