RIPA buffer

Originally designed for isolating proteins from cells for use in RadioImmunoPrecipitation Assays, RIPA buffer is a good general-purpose whole-cell protein extraction method useful for screening cells by WesternBlotting. The ease of use of this method of protein isolation offsets the disadvantage of not giving a particularly "pure" protein extract (i.e. there will be significant levels of non-protein contaminants). If high purity is important, more elaborate protocols are called for.

RIPA buffer:

• 50 mM Tris-HCl, pH 7.4
  
• 150 mM NaCl
  
• 1% NP-40 (dilute from a 10% stock solution in water)
  
• 1% deoxycholate (dilute from a 10% stock solution of Na-deoxycholate in water → protect from light)
  
• 0.1% SDS
  
• 1 mM EDTA (diluted from a 500 mM, pH 8.0 stock solution in water)
  
• protease inhibitors¹
  
  • to be added immediately before use
    

Store RIPA buffer at 4C.

¹protease inhibitors can be added individually (eg, PMSF (phenylmethylsulfonylfluoride), aprotinin, leupeptin, pepstatin etc) or from a commercially available "protease inhibitor cocktail". The cocktail method is generally more successful, much easier to use and worth the additional monetary cost involved.

Basic protocol:

1. isolate tissue culture cells.
   
   • for adherent cells:
     
     1. aspirate medium and treat with trypsin-EDTA to detatch the cells from the plate
        
     2. add two volumes of complete medium with serum to the trypsin-EDTA treated cells, pipette up and down to break up clumps and transfer to a 15 mL centrifuge tube
        
     3. pellet cells in a clinical centrifuge (500 g for 5 minutes)
        
     4. aspirate the supernatant
        
     5. resuspend cells in complete medium containing serum (to inactivate the trypsin)
        
     6. pellet cells again and aspirate supernatant
        
   • for suspension cells:
     
     1. transfer cells to a 15 mL centrifuge tube, pellet cells in a clinical centrifuge (500 g for 5 minutes), aspirate the supernatant
        
2. resuspend the cell pellet in 5 mL ice-cold PBS
   
3. pellet in a clinical centrifuge (500 g for 5 minutes)
   
4. aspirate the PBS supernatant
   
5. resuspend the cell pellet by gentle trituration with a pipette or pipette tip (depending on volume) in 1 ml RIPA buffer (with added protease inhibitors) per 5x10⁶ cells, transfer to a 1.5 ml microcentrifuge tube
   
6. pellet the cellular debris by centrifugation in a microcentrifuge for 5 minutes at maximum speed (at 4C if possible)
   
7. remove the supernatant to a new microcentrifuge tube on ice
   
   • optional (strongly encouraged): measure the protein concentration of the supernatant at this stage
     
8. add ¹/₅^th volume (200 μl) 6x SDS sample buffer, mix thoroughly and place in a boiling water bath for 2 minutes
   
9. store at -20C
   

Approximate whole cell protein yields from 5x10⁶ cells per 1 ml RIPA buffer (subject to wide variation)

HeLa	3 mg/ml
GM00637	1.5 mg/ml

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