SPECTROFLUOR® uPA, fluorogenic substrate for uPA assays

Description
Fluorogenic substrate for uPA.

Chemistry
Formula: Glutaryl-Gly-Arg-AMC (C23H30N6O7)
Chemical name: Glutaryl- glycyl- L-arginine- 4-methylcoumaryl- 7-amide
MW: 502.53 Dalton
Purity: >98% by HPLC
Absorption: Maximum Wavelength 380[ex] nm
Emission: Maximum Waelength 460[em] nm

Principle
 
The initial rate of increase in the AMC concentration can be monitored fluorometrically at 380_[ex] nm and
460_[em] nm.

Assay conditions
Enzyme activity is determined by measuring the increase in absorbance of the free chromophore (AMC) generated, in comparison to the original substrate, per unit time 460_[em] nm. At excess substrate concentration, the rate of absorbance increase due to the amount of chromophore released is linearly related to enzyme concentration.
Measurement can be made either through acid quenching of the reaction (a. end-point method), or through use of a kinetic recording spectrophotometer (b. initial-rate-method).

Procedure
Choose the proper conditions for the measurement, such as substrate, enzyme concentration and other reaction conditions, depending on the purpose of the measurement.
1. Set the fluorescence plate reader at 380_[ex] nm, 460_[em] nm at 25 °C. (1.0 relative fluorerscence
    unit at 10^-6 M of AMC).
2. Pipette 160 µl of buffer and 20 µl of substrate (1 mM in DMSO) in the wells of a 96-well plate
    (for final concentration of 100 µM).
3. Incubate the plate in the fluorescence plate reader for 3-4 min (for temperature equlibration).
4. Take the 96-well plate out and add 20 µl enzyme solution in each well.
5. Mount the plate in the fluorescence plate reader.
6. Record the increase in fluorescence intensity for 30 min with a premixing time of 3 sec.
7. Calculate the amount of released.

Presentation
The vial contains 5 mg (=10 μmol) substrate, which has been lyophilized as an amorphous powder from acetic acid solution.
Due to the nature of the material, it may coagulate at the bottom of the vial to form resinous droplets during storage. However, this kind of appearance change is not related to any deterioration in the guaranteed quality.
Reconstitution
Carefully open the cap, add 1 ml DMSO to create a 10 mM solution. Close the cap tightly, and shake well until all the contents are dissolved.

Storage
May be used by the expiry date given on the label when stored unopened, protected from moisture in the dark at 2-8 °C.
After reconstitution in DMSO, the solution is stable for more than one month if it is protected from moisture and kept in the dark at -20 °C.

Category: Research use only

Type: Substrate

Product Availability: Worldwide

Manufacturer: ImmBioMed GmbH & Co KG, Germany

For more information please click .pdf icon below.