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Protokoll
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A. General Consideration & Reagent Preparation: Reconstitute the lyophilized Active β -Secretase to 10 μ l ddH 2 O. The enzyme should be refreezed immediately at –70 o C after each use to avoid loss of activity. The enzyme is sufficient for 5 positive control assays (2 μ l/assay). Assay can be performed directly in a 96-well plate. Nunc F16 Black MaxiSorp TM polystyrene microplate is recommended. B. 1. Collect cells (5 x 10 6 cells/assay) by centrifugation for 5 min at 700x g. Add 0.1 ml of ice-cold Extraction Buffer. For tissue sample, add 2-3X volume of ice-cold Extraction Buffer to tissue sample and homogenize it on ice. 2. Incubate cell lysate on ice for 10 minutes and centrifuge at 10,000x g for 5 minutes. Transfer the supernatant to a new tube and keep on ice. This should yield a lysate with a protein concentration of ~2-4 mg/ml. 3. Add 50 μ l cell lysate (~2 - 5 x 10 6 cells or 25 - 200 μ g of total protein) to each well in a 96-well plate. For positive control assay, add 2 μ l of reconstituted Active β -secretase to 50 μ l of Extraction Buffer. For negative control assay, add 2 μ l of the β -Secretase Inhibitor to 50 μ l of Extraction Buffer. 4. Add 50 μ l of 2X Reaction Buffer. 5. Add 2 μ l of β -Secretase substrate. 6. Cover the plate, tap gently to mix, and incubate in the dark at 37 o C for 1-2 hours. 7. Read sample in a fluorescence plate reader with Ex. = 335-355 nm and Em. = 495-510 nm. Background reading from substrate (without secretase) must be subtracted from all treated and untreated samples before calculating the fold increase in secretase activity (Note: Background reading from substrate can be quite high, due to the nature of such fluorescence quenching assay.) β -Secretase activity can be expressed as the Relative Fluorescence Units per μ g of protein sample.
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