Veritas™
Microplate Luminometer Method for
Alkaline Phosphatase
1. INTRODUCTION
The Turner BioSystems' Veritas™ Microplate
Luminometer has a large dynamic range that is highly suited for alkaline
phosphatase assays. Alkaline phosphatase is a stable enzyme often conjugated
to secondary antibodies for immunoassays. Alkaline phosphatase is also
a useful genetic reporter and a helpful tool in DNA cloning.
The superior sensitivity of the Veritas permits
detection of very low levels of alkaline phosphatase activity. The Veritas
can detect as little as 3 x 10-21 moles alkaline phosphatase
enzyme. Measurements are linear over four orders of magnitude (Figure
1). All tests were conducted using Lumigen's
APS-5 (Lumigen, Southfield, MI) and purified calf intestinal mucosa
alkaline phosphatase (Biozyme, San Diego, CA).
Figure 1: Detection of alkaline phosphatase
with the Veritas™ Microplate Luminometer. 90 µL APS-5 was added
to 10 µL alkaline phosphatase diluted in dH2O. Samples
were incubated at room temperature for 5 minutes before measurement.
2. MATERIALS REQUIRED
- Veritas™ Microplate Luminometer (P/N
9100-000)
- 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 Veritas icon
to start the software.
3.2.2 Click on "Create New Protocol"
from the "Welcome to Veritas" dialog box.
3.2.3 Using the new protocol wizard, set
up a protocol with 0 injectors, 1 second integration and 5 minute delay.
Select the wells you wish to read and click on "Finish".
3.2.4 Enter your information into the "Experiment",
"Operator", "Plate No.", and "Notes" fields
in the "Main Dialog Box".
3.3 Sample Analysis
3.3.1 Equilibrate alkaline phosphatase,
samples and APS-5 to room temperature.
3.3.2 Prepare a 10-fold 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 Insert the plate into the Veritas
and click on "Start" to begin assay. After the programmed
5-minute delay, the Veritas will begin measurement. RLU values measured
by the Veritas will appear in the Excel spreadsheet after all the selected
wells in each row have been read. If you encounter an error message,
refer to the troubleshooting guide in the Veritas Operating Manual for
more information.
Note: It is not recommended to open
another Excel spreadsheet while the Veritas reads your sample plate.
3.3.6 Once the measurements are complete,
you can access Excel to analyze your data.
3.3.7 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
Veritas is a trademark of 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
www.turnerbiosystems.com
Mailing Address:
Turner BioSystems, Inc.
645 N. Mary Avenue
Sunnyvale, CA 94085
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