Purpose of method:  extraction of expansin protein from native plant cell walls

1. Use at least 10 g FW tissue from the growing region (it's OK to include some tissue that has just ceased growing - often it has residual expansin activity). We generally collect 50-100 g of cucumber hypocotyls for our extractions.

2. In a chilled Waring blender (or a chilled mortar and pestle if you decide to use much less than 10 g of tissue), homogenize well in homogenization buffer. 20 s to 60 s at medium-high speed is what we generally use, depending on the toughness of the tissue. Look at the tissue fragments under a microscope to assess how well disrupted the cells are. Use a ratio of 10 mL of buffer per 1 g of tissue.

3. The homogenization buffer is 25 mM HEPES, 2 mM sodium metabisulfite (reducing agent), 2 mM EDTA (metal chelator), brought to pH 7.0 with NaOH. Often we include 0.1% Triton X-100 in the buffer. This yields a cleaner wall protein prep, but leads to foaming during homogenization, and must be WASHED out WELL from the walls before extraction. The foaming is usually troublesome during the filtration and washing step. I would suggest you omit the Triton in the first run, since it does not seem to help in activity assays. If you don't have Hepes, TRIS ought to work fine.

4. Collect the walls on a filter. We use a Spectra/Mesh nylon mesh with 70-um holes. Miracloth also works well, but is more fragile. Wash with copious amounts of homogenization buffer or chilled distilled water (we use the latter to save time and materials). Wash until the wash solution look clean. Then squeeze the walls in the filter to remove excess water (close up the top of the filter by twisting/clamping with your gloved hand and GENTLY, slowly apply pressure to force out the water).

5. Add extraction buffer to wet walls. We use enough buffer make a loose slurry, like wet oatmeal. The ratio of buffer to walls depends on the wall characteristics, how well you removed the water from the walls, etc. I would estimate 3 mL buffer for each g squeezed walls.

6. The extraction buffer is 1 M NaCl, 25 mM HEPES, 2 mM EDTA, 2 mM sodium metabisulfite, brought to pH 7.0 with NaOH. Sometimes we find that a 1-2 h pre-incubation in the homogenization buffer at room temperature before extraction helps to increase the extractability of expansins. The reason for this is not certain, but we suspect the wall is being modified by endogenous enzymes. It may also have to do with hydration and dissolution of wall components.

7. We let the walls extract in a 4-degree cold room on an agitator to keep the slurry in gentle motion. Avoid stir bars, which get stuck and often generate foaming (=protein denaturation). 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.

8. Filter the wall slurry as before on a mesh or Miracloth. Re-extract a second time, if desired.

9. 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. Pellet the precipitate by centrifugation (5,000g for 10 min is generally sufficient; high speed on a table top centrifuge also works). 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.