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now: Thu Jun 24 07:33:41 2021 ... mod: Wed Mar 24 19:44:13 2021
Originally designed for isolating proteins from cells for use in R
ssays, 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.
- 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.
- isolate tissue culture cells.
- for adherent cells:
- aspirate medium and treat with trypsin-EDTA to detatch the cells from the plate
- 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
- pellet cells in a clinical centrifuge (500 g for 5 minutes)
- aspirate the supernatant
- resuspend cells in complete medium containing serum (to inactivate the trypsin)
- pellet cells again and aspirate supernatant
- for suspension cells:
- transfer cells to a 15 mL centrifuge tube, pellet cells in a clinical centrifuge (500 g for 5 minutes), aspirate the supernatant
- resuspend the cell pellet in 5 mL ice-cold PBS
- pellet in a clinical centrifuge (500 g for 5 minutes)
- aspirate the PBS supernatant
- 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
- pellet the cellular debris by centrifugation in a microcentrifuge for 5 minutes at maximum speed (at 4C if possible)
- remove the supernatant to a new microcentrifuge tube on ice
- optional (strongly encouraged): measure the protein concentration of the supernatant at this stage
- add 1/5th volume (200 μl) 6x SDS sample buffer, mix thoroughly and place in a boiling water bath for 2 minutes
- 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|