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now: Wed May 4 17:14:37 2016 ... mod: Thu Mar 20 09:44:02 2014
VandyBren |I understand that you want more of this peorcss automated, but you don't really give any ideas as to how to automate it or even which parts of the peorcss you have in mind for automation. I do not quite grasp how you would automate any of these steps to a point where you would need any less human input. I suppose some of the calculations could be automatic, but by the time you run that through an automated system you probably could have just calculated it yourself.
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|