IGEM:IMPERIAL/2009/Assays Protocols/Trehalose

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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:

Other Trehalose Testing Kits:

Sigma's glucose test kit

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)