Purpose of method: Reconstitution assay for expansin

Detailed method:

1. Inactivate endogenous expansins in the wall samples:
   
   Take the growing regions to be tested, freeze, thaw and abrade the cuticle with washed carborundum slurry. Rinse off the carborundum. Place the walls in 95 degree C water for 15 min. You may have to vary the time and temperature for your tissue. We have tried various treatments such as 15 s dip in boiling water, 85 degrees for 10 min, etc. We find that the heat treatment needed to kill endogenous activity varies with time, and we don't know why. If you don't kill the endogenous activity, your baselines will be too high. Other treatments that have worked are 4 h Pronase treatment (followed by washing to remove the protease) and 4 h extraction in 1% SDS (followed by wash).
   

2. Press the walls to flatten them and remove water (they clamp better this way). Hang the inactivated walls in 50 mM acetate buffer, pH 4.5, at 20 g tension (vary this according to your walls) until creep rate stabilizes (20-30 min, generally).

3. Exchange the buffer for the same buffer containing the protein to be tested.

4. To test the creep activity in the crude ammonium sulfate wall pellet (see extraction protocol), dissolve the pellet in enough 4.5 acetate buffer that salt levels are below 300 mOsm/kg (test in a Wescor vapor pressure osmometer). Our protein pellets are in the bottom of a 50-mL round centrifugation tube, and have an estimated volume equivalent of about 2 cooked rice grains (long-grain Basmati type, mashed). We use 5-7 mL of buffer to dissolve such a pellet. If you use too little buffer, the salt levels may be too high and interfere with the activity assay. Too much buffer and the protein may be too dilute. You have to do some trial and error. Also, the proteins may be desalted and concentrated on a Centricon 30 kD membrane centrifuge system (prefilter on a 0.2 or 0.45 um filter remove particulates before filtering on the 30 kD filter. Otherwise, the filter will clog).

5. CHECK and ADJUST pH BEFORE ASSAYING.