GloRunner™
Microplate Luminometer Method for
Alkaline Phosphatase
1.
INTRODUCTION
The Turner
BioSystems GloRunner™ Microplate Luminometer in combination with
the APS-5 substrate from Lumigen, provides a quick, reliable method for
measuring alkaline phosphatase activity. Alkaline phosphatase is a stable
enzyme often conjugated to secondary antibodies for immunoassays. Alkaline
phosphatase is also a useful genetic reporter.
The GloRunner
detects as little as 3 x 10-20 moles alkaline phosphatase.
Measurements are linear for over three orders of magnitude (Figure 1).
All tests were conducted using Lumigen
APS-5 (Lumigen, Southfield, MI) and purified calf intestinal mucosa
alkaline phosphatase (Biozyme, San Diego, CA).
Figure 1: Detection of alkaline phosphatase with the GloRunner™
Microplate Luminometer.
90 µL APS-5 was added to 10 µL allkaline phosphatase diluted
in dH2O. Samples were incubated at room temperature for 5 minutes before
measurement.
2. MATERIALS
REQUIRED
- GloRunner™
Microplate Luminometer (P/N 9000-000)
- 96-well
plates, white (E&K Scientific EK 25075)
- Lumigen
APS-5
- Alkaline
phosphatase (Biozyme ALPI12G)
- p200 pipette
and pipette tips
- p20 pipette
and pipette tips
3. EXPERIMENT
PROTOCOL
3.1 Reagent Preparation
- Lumigen
APS-5: Use as supplied. Store at 2–8°C, where it is stable
for up to 12 months.
- Alkaline
phosphatase: Store at 4°C.
3.2 Instrument
Setup
3.2.1 Double-click
on the GloRunner icon to start the software.
3.2.2 Open
a new workbook and select the wells you wish to read from the "Options"
screen.
3.2.3 You
may also wish to select a delay for incubation, the number of repeated
runs, and a delay before microplate ejection. The recommended measurement
duration is 1 second.
3.3 Sample
Analysis
3.3.1 Equilibrate
alkaline phosphatase, samples and APS-5 to room temperature.
3.3.2 Prepare
a serial dilution of alkaline phosphatase in your assay buffer.
3.3.3 Add
10 µL of sample or standard to the 96-well plate.
3.3.4 Add
90 µL of APS-5 reagent to the sample or standard.
Note: The recommended ratio of sample volume to APS-5 volume is 1:10 and should
not exceed 1:1
3.3.5 Incubate
the microplate for 5 minutes at room temperature.
3.3.6 Insert
the plate into the GloRunner sample tray and click on "Start"
to begin assay.
3.3.7 The
data will appear in the Excel spreadsheet after the GloRunner completes
the run.
3.3.8 Please
make sure to remove your plate after measurement.
4. ABOUT
LUMIGEN
Orders
for Lumigen's products may be placed by:
Phone: (248) 351-5600
Fax: (248) 351-0518
E-mail: marketing@lumigen.com
Internet: www.lumigen.com
Mailing address:
Lumigen, Inc.
22900 West Eight Mile
Southfield, MI 48034
USA
5. ABOUT
BIOZYME
Orders for
Biozyme's products may be place by:
Phone:
(800) 423-8199
Fax: (858) 549-0138
E-mail: bioinfo@biozyme.com
Internet: www.biozyme.com
Mailing
address:
Biozyme Laboratories International Ltd.
9939 Hibert Street, Suite 101
San Diego, CA 92131
6.
ABOUT TURNER BIOSYSTEMS
Orders
for Turner BioSystems' products may be placed by:
Phone: (408)
636-2400
Toll Free: (888) 636-2401 or
Fax: (408) 737-7919
Contact us
via our contact form
Internet: www.turnerbiosystems.com
Mailing Address:
Turner BioSystems, Inc.
645 N. Mary Avenue
Sunnyvale, CA 94085
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