LeBauer:Protocol/Enzyme: Difference between revisions
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==Materials== | ==Materials== | ||
===Solutions=== | |||
*1.0 M NaOH [[Sodium hydroxide]] | *1.0 M NaOH [[Sodium hydroxide]] | ||
** 4g NaOH pellets | ** 4g NaOH pellets | ||
| Line 19: | Line 19: | ||
**fill 1000 ml volumetric flask | **fill 1000 ml volumetric flask | ||
**titrate solution to pH = 5 with additional acetic acid | **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>\beta</math>-glucosidase===== | ||
** | *5 mM p-Np-<math>\beta</math>-gucopyranoside | ||
** | **150.7 mg substrate | ||
* | **100 ml buffer | ||
* | * 100 ml volumetric flask | ||
=====**[https://webfiles.nacs.uci.edu/allisons/public/Protocols/ColorimetricEnzymeAssays.pdf Additional Substrates]===== | |||
===Waste Disposal / Plate Washing=== | |||
==Procedure== | ==Procedure== | ||
| Line 32: | Line 44: | ||
====Preparing assay trays:==== | ====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 [https://webfiles.nacs.uci.edu/allisons/public/Protocols/ColorimetricEnzymeAssays.pdf)]] | |||
#Rinse trays free from any contaminates or debris with sodium acetate buffer. Rinse trays out again with ethanol. Place upside down to dry. | #Rinse trays free from any contaminates or debris with sodium acetate buffer. Rinse trays out again with ethanol. Place upside down to dry. | ||
#Label one set of trays BG (A-H) and another set PPO (A-H) | #Label one set of trays BG (A-H) and another set PPO (A-H) | ||
# | #See figure on right for arrangement of a 96 well plate with 7 reps each of 6 samples per tray. | ||
# | |||
====Combining enzymes and substrates:==== | |||
#Add 200μl buffer to blank wells | |||
#Add 150μl buffer to homogenate control wells | |||
#Add 50μl buffer to substrate control wells | |||
#Add 50 µl homogenate to the homogenate control and assay wells. | |||
#Add 150 µl substrate to the substrate control and assay wells. | |||
See [2] for more details. Analysis on spectrometer follows Allison's [https://webfiles.nacs.uci.edu/allisons/public/Protocols/ColorimetricEnzymeAssays.pdf procedure]. | |||
==Notes== | ==Notes== | ||
| Line 45: | Line 67: | ||
==References== | ==References== | ||
'''Relevant papers | '''Relevant papers''' | ||
# Allison, S.D., and Jastrow, J.D. 2006. Activities of extracellular enzymes in physically isolated fractions of restored grassland soils [[DOI:10.1016/j.soilbio.2006.04.011]] | |||
# LeBauer, D.S. 2010. Litter degradation rate and beta-glucosidase activity increase with fungal diversity. Canadian Journal of Forest Research. 40(6): 1076–1085 [[DOI:10.1139/X10-054]] | |||
[[Category:Protocol]] | [[Category:Protocol]] | ||
[[Category: | [[Category:Enzyme]] | ||
Latest revision as of 09:16, 9 July 2010
Overview
Enzyme assays for litter and soil.
Adapted from
- Steven Allison 2008 (Allison Lab),
- Bob Sinsabaugh 1994 (Center for Dead Plant Studies)
Materials
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
**Additional Substrates
Waste Disposal / Plate Washing
Procedure
Preparing soil sample:
- Add 10 ml sodium acetate buffer into each soil tube. Shake gently.
- Place tubes on plate shaker for 1 hour on speed 180 for mixing.
Preparing assay trays:
- Rinse trays free from any contaminates or debris with sodium acetate buffer. Rinse trays out again with ethanol. Place upside down to dry.
- Label one set of trays BG (A-H) and another set PPO (A-H)
- See figure on right for arrangement of a 96 well plate with 7 reps each of 6 samples per tray.
Combining enzymes and substrates:
- Add 200μl buffer to blank wells
- Add 150μl buffer to homogenate control wells
- Add 50μl buffer to substrate control wells
- Add 50 µl homogenate to the homogenate control and assay wells.
- Add 150 µl substrate to the substrate control and assay wells.
See [2] for more details. Analysis on spectrometer follows Allison's procedure.
Notes
- List troubleshooting tips here.
- You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
- Anecdotal observations that might be of use to others can also be posted here.
Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.
References
Relevant papers
- Allison, S.D., and Jastrow, J.D. 2006. Activities of extracellular enzymes in physically isolated fractions of restored grassland soils DOI:10.1016/j.soilbio.2006.04.011
- LeBauer, D.S. 2010. Litter degradation rate and beta-glucosidase activity increase with fungal diversity. Canadian Journal of Forest Research. 40(6): 1076–1085 DOI:10.1139/X10-054
