7. Quality Control
7.3 Glow Type Luminescence
7.3.1 Sensitivity
Test
•
Use a white Greiner 96 well plate and the black cover plate, which is
supplied with the instrument.
•
Prepare a 1:1 dilution series of alkaline phosphatase (molecular biology
grade source: Boehringer) in assay buffer (20 mM Tris HCl pH 9.8, 1 mM
MgCl
2
; source: Tropix). Highest concentration for measurement of sensitivity
may be 10
-6
DEA activity units / µl.
•
Apply 20 µl of pure buffer to wells A1,...,A4;...;D1,...,D4 these will serve as
blanks. Continue applying 20 µl of increasing AP dilution to the next columns
in replicates of 4.
•
Finally add 100 µl of chemiluminescent substrate (CDP-Star with Emerald-II
enhancer, source: Tropix) per well and mix thoroughly. Incubate at room
temperature for 20 minutes.
•
Integrate 2 seconds per well using gain 100. Chemiluminescent signal will
change very slowly over the following hour when using the above maximum
enzyme concentration.
The procedure to achieve the sensitivity is as follows:
Plot the blanked values of the dilution series against the corresponding activity
units and calculate a linear fit. Divide the standard deviation of the blanks by the
slope of the fitting curve. Multiply with a factor of 2 to achieve statistical reliability.
The sensitivity is expected to be <0.4 10
-6
DEA activity units/well
7-4
Operating Manual for GENios, GENios FL, and GENios
Plus
No: I 112 904 Rev No: 1.1
June 2002
