IGEM:IMPERIAL/2009/Assays Protocols/Trehalose: Difference between revisions
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===Trehalose=== | ===Trehalose=== | ||
Trehalose will be produced when functional OtsA and OtsB are both present and expressed. | |||
A good trehalose assay kit appears to be the Megazyme Trehalose assay kit: | |||
Info: | |||
http://secure.megazyme.com/downloads/en/data/K-TREH.pdf | |||
Purchase: | |||
http://secure.megazyme.com/Dynamic.aspx?control=CSViewProduct&categoryName=AssayKits&productId=K-TREH | |||
Quantitative Assay: | |||
[[Image:Measure3.JPG]] | |||
[[Image:Measure4.JPG]] | |||
Other Trehalose Testing Kits: | |||
[http://www.biokits.com/moreinfos.html?id=1266 Sigma's glucose test kit] | [http://www.biokits.com/moreinfos.html?id=1266 Sigma's glucose test kit] | ||
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alternatively, use [http://www.biokits.com/moreinfos.html?id=360 sigma's trehalose test kit] | alternatively, use [http://www.biokits.com/moreinfos.html?id=360 sigma's trehalose test kit] | ||
=== Individual Assays === | === Individual Assays === | ||
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Quantative testing of each enzyme is tested using the following assays: | Quantative testing of each enzyme is tested using the following assays: | ||
[[IGEM:IMPERIAL/2009/Assays_Protocols/Trehalose/OtsA | OtsA]] | |||
[[IGEM:IMPERIAL/2009/Assays_Protocols/Trehalose/OtsA | OtsA]] (Trehalose–6-Phosphate Synthase) | |||
* quantitative assay - giving units of activity for the enzyme.<br> | |||
* By measuring OD2340 <br> | |||
Final output= nmol UDP/min/mg protein <br> | |||
<br> | |||
[[IGEM:IMPERIAL/2009/Assays_Protocols/Trehalose/OtsB | OtsB]] (Trehalose–6-Phosphate Phosphatase) |
Latest revision as of 03:49, 5 August 2009
Trehalose
Trehalose will be produced when functional OtsA and OtsB are both present and expressed.
A good trehalose assay kit appears to be the Megazyme Trehalose assay kit:
Info:
http://secure.megazyme.com/downloads/en/data/K-TREH.pdf
Quantitative Assay:
Other Trehalose Testing Kits:
Reference paper
1. Extract trehalose by boiling cell pellets at 95degrees for 20mins.
2. Convert trehalose in the supernatant to glucose with trehalase obtained from sigma.
3. Measured this by a glucose assay kit from sigma.
4. Determine the preexisting glucose in each sample was determined in a control reaction without trehalase
5. Subtracted this from total glucose
alternatively, use sigma's trehalose test kit
Individual Assays
Trehalose is produced after conversion from 2 enzymes, coded for by OtsA and OtsB. These are Trehalose phosphate synthase and Trehalose phosphate phosphatase respectively.
Quantative testing of each enzyme is tested using the following assays:
OtsA (Trehalose–6-Phosphate Synthase)
- quantitative assay - giving units of activity for the enzyme.
- By measuring OD2340
Final output= nmol UDP/min/mg protein
OtsB (Trehalose–6-Phosphate Phosphatase)