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Team:TU Darmstadt/Protocols/pNP Assay
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Method: Measurement of absorption at 405nm every minute over 30 minutes, after addition of enzyme. The released pNP-group has a typical absorbtion at 405nm wavelength. The absorbtion is meassured by an ELISA reader capable of 96 well plates. | Method: Measurement of absorption at 405nm every minute over 30 minutes, after addition of enzyme. The released pNP-group has a typical absorbtion at 405nm wavelength. The absorbtion is meassured by an ELISA reader capable of 96 well plates. | ||
| - | [[File:pnpb_spalt.png|thumb|center|The mechanism of pNPB degradation. The release of pNP is meassured by the characteristic absorption at 405 nm]] | + | [[File:pnpb_spalt.png|thumb|center|600px|The mechanism of pNPB degradation. The release of pNP is meassured by the characteristic absorption at 405 nm]] |
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==== Reagents ==== | ==== 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 /> | + | * A 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 /> | + | * B 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 /> | * 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-3-phenylpropanoate: 27mg in methanol diluted to a concentration of 1mM<br /> | |
| - | * Enzyme stock solution<br /> | + | * C Enzyme stock solution<br /> |
| - | + | ||
==== 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 | + | #: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. | ||
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==== Reagents ==== | ==== 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 /> | + | * A 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 /> |
| - | *4-Nitrophenyl butyrate: 8.8 µL in 1mL acetonitril diluted to a concentration of 10mM<br /> | + | * B 4-Nitrophenyl butyrate: 8.8 µL in 1mL acetonitril diluted to a concentration of 10mM<br /> |
| - | *Bacteria with different concentrations of arabinose for induction (0.5%, 0.2%, 0.02% and 0%) | + | * C Bacteria with different concentrations of arabinose for induction (0.5%, 0.2%, 0.02% and 0%) |
==== Procedure ==== | ==== Procedure ==== | ||
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#100µL of these four suspensions are added to several wells of grainer 96 well plate<br /> | #100µL of these four suspensions are added to several wells of grainer 96 well plate<br /> | ||
#To these 10µL of diluted reagent B are pipetted<br /> | #To these 10µL of diluted reagent B are pipetted<br /> | ||
| - | #From this moment on each minute to a total count of 30 minutes the respective suspensions' absorptions is recorded | + | #From this moment on each minute to a total count of 30 minutes the respective suspensions' absorptions is recorded |
Latest revision as of 00:44, 27 September 2012
Contents |
pNP-Assay
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. The released pNP-group has a typical absorbtion at 405nm wavelength. The absorbtion is meassured by an ELISA reader capable of 96 well plates.
Enzyme
Reagents
- A 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
- B 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-3-phenylpropanoate: 27mg in methanol diluted to a concentration of 1mM
- C 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.
Bacteria
Reagents
- A 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
- B 4-Nitrophenyl butyrate: 8.8 µL in 1mL acetonitril diluted to a concentration of 10mM
- C Bacteria with different concentrations of arabinose for induction (0.5%, 0.2%, 0.02% and 0%)
Procedure
- A bacteria suspension with a OD of 0.1 is prepared from each bacterial culture induced by different arabinose concentrations (reagent C) in reagent A
- 100µL of these four suspensions are added to several wells of grainer 96 well plate
- To these 10µL of diluted reagent B are pipetted
- From this moment on each minute to a total count of 30 minutes the respective suspensions' absorptions is recorded
