"
Team:Washington/Protocols/Purified Enzyme Assay
From 2011.igem.org
(Difference between revisions)
(→Whole Cell Lysate Assay) |
|||
| (3 intermediate revisions not shown) | |||
| Line 2: | Line 2: | ||
| - | = | + | =Enzyme Assay= |
<html> | <html> | ||
| Line 28: | Line 28: | ||
</html> | </html> | ||
<!---------------------------------------PAGE CONTENT GOES BELOW THIS----------------------------------------> | <!---------------------------------------PAGE CONTENT GOES BELOW THIS----------------------------------------> | ||
| + | |||
| + | ==Substrate== | ||
| + | The substrate was ordered at a 20% discount from Anasepc in support of Undergraduate education. The peptide Motif PQPQLP commonly found in gluten was synthesized with a fluorophore (5-FAM) or Quencher (QXL52) available from anaspec. It is denoted as the following | ||
| + | |||
| + | Ordered Peptide (95% Purity): QXL520-PQPQLP-K(5-FAM)-NH2 | ||
| Line 35: | Line 40: | ||
***Use the P20 multichannel with a taped cardboard stopper to make sure you don't hit the pellet! | ***Use the P20 multichannel with a taped cardboard stopper to make sure you don't hit the pellet! | ||
**Monitor the reaction with the SpectraMax | **Monitor the reaction with the SpectraMax | ||
| + | ***SpectraMax Settings: | ||
| + | ****Excitation: | ||
| + | ****Emission: | ||
| + | ****Cutoff: | ||
Latest revision as of 23:16, 28 September 2011
Enzyme Assay
Substrate
The substrate was ordered at a 20% discount from Anasepc in support of Undergraduate education. The peptide Motif PQPQLP commonly found in gluten was synthesized with a fluorophore (5-FAM) or Quencher (QXL52) available from anaspec. It is denoted as the following
Ordered Peptide (95% Purity): QXL520-PQPQLP-K(5-FAM)-NH2
- Assay
- Add 90uL of 5uM substrate (in NaAc pH 4.0 buffer) to each well of a black fluorescent microtiter assay plate
- Start reaction by adding 0.0125mg/mL enzyme (try to avoid bubbles and pippette quickly, but accurately)
- Use the P20 multichannel with a taped cardboard stopper to make sure you don't hit the pellet!
- Monitor the reaction with the SpectraMax
- SpectraMax Settings:
- Excitation:
- Emission:
- Cutoff:
- SpectraMax Settings:
