LeBauer:Protocol/Enzyme

Overview

Enzyme assays for litter and soil.

Adapted from

• Steven Allison 2008 (Allison Lab),
  • Steve's Protocol
• Bob Sinsabaugh 1994 (Center for Dead Plant Studies)

Materials

List reagents, supplies and equipment necessary to perform the protocol here. For those materials which have their own OWW pages, link to that page. Alternatively, links to the suppliers' page on that material are also appropriate.

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

Substrates

PPO polyphenol oxidase

• 50 mM pyrogallolX μL reagent 2
  • 631 mg pyrogallol (CAS 87-66-1)
  • 1.861 g disodium dihydrate EDTA
  • 100 ml buffer
• 100 ml volumetric flask

BG: [math]\displaystyle{ \beta }[/math]-glucosidase

• 5 mM p-Np-[math]\displaystyle{ \beta }[/math]-gucopyranoside
  • 150.7 mg substrate
  • 100 ml buffer
• 100 ml volumetric flask

Safety

Gloves. Lab coat. Splash goggles. Respirator or Fume hood

Procedure

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:

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. For 6 samples per tray, with 7 reps each, use the following arrangement:
   • (Also see Steve's layouts of 5 or 7 samples per tray [1])

1. 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.

Notes

1. List troubleshooting tips here.
2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
3. Anecdotal observations that might be of use to others can also be posted here.

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References

Relevant papers and books

1. Goldbeter A and Koshland DE Jr. An amplified sensitivity arising from covalent modification in biological systems. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6840-4. DOI:10.1073/pnas.78.11.6840 |
2. JACOB F and MONOD J. Genetic regulatory mechanisms in the synthesis of proteins. J Mol Biol. 1961 Jun;3:318-56. DOI:10.1016/s0022-2836(61)80072-7 |
3. ISBN:0879697164

Contact

• Who has experience with this protocol?

or instead, discuss this protocol.