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RipaBuffer
RipaBuffer

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:

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 1/5th 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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