Biotek PowerWave, Operator'S Manual

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PowerWave Operator’s Manual 4-15
Linearity Test
The linearity of the reader also can be tested by preparing a series of solutions
of varying absorbances.
The absorbance of a solution is a function of how much light is absorbed by
that solution when it contains a colored compound. A solution that has twice
as much color as another solution will have twice the optical density. (Any
solution that gives a stable color will suffice. This includes substrates
incubated with an enzyme preparation and then stopped with an acidic or
basic solution.) Some enzyme/substrate combinations are given in Table 4-1.
Table 4-1 :
Typical Enzyme-Substrate Combinations and Stopping Solutions
Enzyme Substrate Stopping Solution
Alkaline Phosphate o-nitrophenyl phosphate 3N sodium hydroxide
beta-Galactosidase o-nitrophenyl -beta-D
galactopyranoside
1M sodium carbonate
Peroxidase ABTS 2, 2 Amino-di-(3 ethyl-
[benzthazolin sulfonate] citrate-phosphate buffer, pH 2.8
Peroxidase o-phenylenediamine 0.03N sulfuric acid
A more basic method is to use food coloring available at supermarkets (e.g.
Durkee
ô
food coloring.)
EQUIPMENT REQUIRED
:
•
Distilled water
•
Pipet
•
Yellow food coloring (Example: Durkee

yellow food coloring, .3
oz. bottle)
•
Flat-bottom microplates (Example: Costar

#3590)
•
Tween

20 (polyoxyethylenesorbitan Monolaurate)
•
Beakers
•
Rack of 10 test tubes (10 ml minimum volume)