LeBauer:Protocol/Enzyme: Difference between revisions
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#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. | ||
[[Image:EnzymePlate.jpg | thumb | right | 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)]] | |||
#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== | ==Notes== |
Revision as of 12:50, 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
- 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
Contact
- Who has experience with this protocol?
or instead, discuss this protocol.