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Fluorescence assay for determination of protease activity

In research and industrial work it is quite often necessary to test whether samples or solutions are free of protease activity. Using commercially available kits e.g. from Molecular Probes Inc., protease activity can be measured with the new fluorometer from PerkinElmer Life Sciences, VICTOR.

VICTOR is a new multitechnology and multilabel counter from Wallac that can measure fluorescent, photometric and luminescent labels as well as DELFIA research assays in microtitration plates. It is an epifluorometer that allows the use of both clear and coloured plates depending on the assay requirements.

DESCRIPTION OF ASSAY

In the reaction protease digests a highly quenched BODIPY FL-casein substrate releasing fluorescent peptides. The trypsin used as standard was diluted in 10 mmol/l Tris-HCl buffer, pH 7.8 of which 100 μl was added to the well.

After addition of 100 μl substrate, 10 μg/ml, diluted in 10 mmol/l Tris HCl buffer, pH 7.8, the plate was incubated overnight at room temperature and then measured.

DETECTION

The detection was done using the factory preset settings for fluorescein label. Excitation was at 485 nm and emission was measured at 535 nm.

RESULTS

Measurement with the new VICTOR fluorometer gives results comparable to the data given by the kit manufacturer.

BENEFITS

Fast and simple sample preparation.

PRODUCTS AVAILABLE

1420-011 VICTOR2 multilabel counter

1420-012 VICTOR2 multilabel counter with TR-fluorescence option

1420-014 VICTOR2 basic with stacker and robotic loading system

1420-015 VICTOR2 with TRF with stacker and robotic loading sy stem

1420-041 VICTOR2V with stacker

OTHER PRODUCTS NEEDED

Trypsin enzyme from bovine pancreas, activity 10700 units/mg (Sigma T-8253)

EnzCheck Protease Assay kit from Molecular Probes Inc. (E-6638) Labsystems black Combiplate (cat.no. 95029490)

REFERENCES

Insert of the EnzCheck Protease Assay Kit from Molecular Probes Inc. (E-6638).


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