"
Team:TU Darmstadt/Protocols/pNP Assay
From 2012.igem.org
(Difference between revisions)
(→About) |
(→About) |
||
| Line 48: | Line 48: | ||
[[File:pnpb_spalt.png]] | [[File:pnpb_spalt.png]] | ||
| + | |||
| + | ==== Reagents ==== | ||
| + | |||
| + | * 1xPBS buffer (1M) pH 7.4 at 34°C (8g of NaCl, 0.2g of KCl, 1.44g of Na<sub>2</sub>HPO<sub>4</sub> and 0.24g of KH<sub>2</sub>PO<sub>4</sub> in 1L dest. H<sub>2</sub>O<br /> | ||
| + | * One of the primarily named substrates in an organic solvent<br /> | ||
| + | * 4-Nitrophenyl butyrate: 8,8µL in 1mL acetonitril and additional dilutions between 5mM and 50µM<br /> | ||
| + | * Bis(4-nitrophenyl) succinate: 36mg in 1mL DMSO diluted to a concentration of 5mM<br /> | ||
| + | * 4-Nitrophenyl (2E)-3-phenylacrylate: 27mg in methanol diluted to a concentration of 1mM<br /> | ||
| + | * Enzyme stock solution<br /> | ||
| + | |||
| + | ==== Procedure ==== | ||
| + | |||
| + | # 1mL of reagent A was added to 10µL of B and mixed by inversion.<br /> | ||
| + | If Bis(4-nitrophenyl) succinate would be added, add 900µL of reagent A to 100µL of reagent B.<br /> | ||
| + | # Now 100µL of the new solution were pipetted into several wells of a grainer 96 well plate.<br /> | ||
| + | # To half the amount of wells xµL of enzyme solution were added to get the desired concentration of the respective enzyme.<br /> | ||
| + | The volume of each enzyme-solution added was 6.6µL of Est13 or 6 µL of FsC respectively. So Est13 had a concentration of 50nM while FsC had a concentration 5nM.<br /> | ||
| + | # The amount of absorption was measured 30 times, each minute one measuring on every filled well was run and recorded.<br /> | ||
=== Enzyme === | === Enzyme === | ||
=== Bacteria === | === Bacteria === | ||
Revision as of 18:25, 24 September 2012
Contents |
pNP-Assay
About
pNP-assays are a common way to quantify hydrolytic activity. We use para-Nitrophenylbutyrate (pNPB) as a substrate. As the catalysts we use the enzymes[http://partsregistry.org/wiki/index.php?title=Part:BBa_K808025 FsC], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808026 pNB-Est13] or our transformed and induced bacteria.
Conditions: T = 34°C pH = 7.4
Method: Measurement of absorption at 405nm every minute over 30 minutes, after addition of enzyme
Reagents
- 1xPBS buffer (1M) pH 7.4 at 34°C (8g of NaCl, 0.2g of KCl, 1.44g of Na2HPO4 and 0.24g of KH2PO4 in 1L dest. H2O
- One of the primarily named substrates in an organic solvent
- 4-Nitrophenyl butyrate: 8,8µL in 1mL acetonitril and additional dilutions between 5mM and 50µM
- Bis(4-nitrophenyl) succinate: 36mg in 1mL DMSO diluted to a concentration of 5mM
- 4-Nitrophenyl (2E)-3-phenylacrylate: 27mg in methanol diluted to a concentration of 1mM
- Enzyme stock solution
Procedure
- 1mL of reagent A was added to 10µL of B and mixed by inversion.
If Bis(4-nitrophenyl) succinate would be added, add 900µL of reagent A to 100µL of reagent B.
- Now 100µL of the new solution were pipetted into several wells of a grainer 96 well plate.
- To half the amount of wells xµL of enzyme solution were added to get the desired concentration of the respective enzyme.
The volume of each enzyme-solution added was 6.6µL of Est13 or 6 µL of FsC respectively. So Est13 had a concentration of 50nM while FsC had a concentration 5nM.
- The amount of absorption was measured 30 times, each minute one measuring on every filled well was run and recorded.
