We let the walls extract in a 4-degree cold room on an agitator to keep the slurry in gentle motion. A 1-h extraction is generally sufficient. We have found that a second extraction usually recovers nearly as much activity as the first. By the third sequence extraction, the activity is diminished. Filter the wall slurry as before on a mesh or Miracloth.
Re-extract a second time, if desired.
Precipitate the wall protein by adding 0.4 g ammonium sulfate for each mL of extract solution. Add the salt slowing while stirring. Let sit an hour at cold temp to let the precipitate form. Decant the solution and store the pellet in a freezer until ready for assays. For activity assays, don't store in an automatic defrost freezer, as the defrost cycles shorten protein life.