LeBauer:Protocol/Enzyme: Difference between revisions
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<!-- If this protocol has papers or books associated with it, list those references here. See the [[OpenWetWare:Biblio]] page for more information. --> | <!-- If this protocol has papers or books associated with it, list those references here. See the [[OpenWetWare:Biblio]] page for more information. --> | ||
<biblio> | <biblio> | ||
http://dx.doi.org/10.1016/j.soilbio.2006.04.011 | |||
#Goldbeter-PNAS-1981 pmid=6947258 | #Goldbeter-PNAS-1981 pmid=6947258 | ||
#Jacob-JMB-1961 pmid=13718526 | #Jacob-JMB-1961 pmid=13718526 | ||
#Ptashne-Genetic-Switch isbn=0879697164 | #Ptashne-Genetic-Switch isbn=0879697164 | ||
</biblio> | </biblio> | ||
# 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:Enzyme]] | [[Category:Enzyme]] |
Revision as of 12:58, 8 June 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.
- 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.
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 and books
-
://dx.doi.org/10.1016/j.soilbio.2006.04.011
- 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 |
- 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 |
- ISBN:0879697164
- 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