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Team:TU Darmstadt/Protocols/pNP Assay
From 2012.igem.org
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==== Procedure ==== | ==== Procedure ==== | ||
| - | # 1mL of reagent A was added to 10µL of B and mixed by inversion. | + | #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.<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 /> | + | #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. | + | #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 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. | + | #The amount of absorption was measured 30 times, each minute one measuring on every filled well was run and recorded. |
=== Enzyme === | === Enzyme === | ||
=== Bacteria === | === Bacteria === | ||
Revision as of 18:27, 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.
- 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.
