LeBauer:Protocol/Enzyme

Enzyme assays for litter and soil.
Adapted from
 * Steven Allison 2008 (Allison Lab),
 * Steve's Protocol
 * Bob Sinsabaugh 1994 (Center for Dead Plant Studies)

Solutions

 * 1.0 M NaOH Sodium hydroxide
 * 4g NaOH pellets
 * 100mL DI water


 * 50 mM sodium acetate buffer (for 1 L)
 * 4.37 g Sodium acetate anhydrous (CAS 127-09-3)
 * 1.1 ml glacial acetic acid (CAS 64-19-7)
 * fill 1000 ml volumetric flask
 * titrate solution to pH = 5 with additional acetic acid

PPO polyphenol oxidase

 * 50 mM pyrogallolX &mu;L reagent 2
 * 631 mg pyrogallol (CAS 87-66-1)
 * 1.861 g disodium dihydrate EDTA
 * 100 ml buffer
 * 100 ml volumetric flask

BG: $$\beta$$-glucosidase

 * 5 mM p-Np-$$\beta$$-gucopyranoside
 * 150.7 mg substrate
 * 100 ml buffer
 * 100 ml volumetric flask

Preparing soil sample:

 * 1) Add 10 ml sodium acetate buffer into each soil tube. Shake gently.
 * 2) Place tubes on plate shaker for 1 hour on speed 180 for mixing.

Preparing assay trays:
[[Image:EnzymePlate.jpg | thumb | right | 300px| alt=Layout for analyzing enzyme activity on seven subsamples from each of six replicate samples. Also see Steve's layouts of five or seven samples per tray ]


 * 1) Rinse trays free from any contaminates or debris with sodium acetate buffer. Rinse trays out again with ethanol. Place upside down to dry.
 * 2) Label one set of trays BG (A-H) and another set PPO (A-H)
 * 3) See figure on right for arrangement of a 96 well plate with 7 reps each of 6 samples per tray.

Combining enzymes and substrates:

 * 1) Add 200μl buffer to blank wells
 * 2) Add 150μl buffer to homogenate control wells
 * 3) Add 50μl buffer to substrate control wells
 * 4) Add 50 µl homogenate to the homogenate control and assay wells.
 * 5) Add 150 µl substrate to the substrate control and assay wells.

See [2] for more details. Analysis on spectrometer follows Allison's procedure.