Lactase Assay: Reagents
[See Hartman, Suskind & Wright, Principles of Genetics Lab Manual, (1965). pp. 52-58.)]Lactase Assay Reagents
The substrate used in the assay of this enzyme is o-nitrophenyl–D galactoside (ONPG), which, upon hydrolysis of the -galactosidic bond, yields galactose and o-nitrophenol, a yellow compound (absorption max = 450 nm) ( CRC Handbook: #p679, Merck Index, #6541). Enzyme activity is proportional to the increase in A450 during incubation.
REAGENTS, MATERIALS AND CALCULATIONS FOR LACTASE ASSAY page 26
0.1 M PO 4 pH 7.0 BUFFER:
For 200 mL, weigh out: 1.0 g KH2PO 4
1.8 g Na 2HPO4
dissolve in 200 mL H 2O, check pH, adjust to 7.0 if nec. with either H 3PO4 or NaOH. Store at 4C.
0.01 M PO 4 pH 7.0 BUFFER : (for suspension and dilution of enzyme)
Q.s. 50 mL of pH 7.0 0.1 M PO 4 buffer to 500 mL with dH 2O.
20 mM o-nitrophenyl–D galactoside (ONPG): (chromogenic substrate)
Weigh out: 602 mg ONPG
dissolve in about 80 mL 0.01 M PO4 buffer, pH 7.0 with swirling and slight warming.
q.s. with buffer to 100.0 mL.
This reagent is unstable over periods of weeks at 4 C. Aliquots may be frozen to stabllize during storage.
REAGENT TO HALT REACTION:
4% K 2CO3 : dissolve 8 g K 2CO3 in 200 mL dH2 O, stir to dissolve.
MATERIALS AND EQUIPMENT for team of four assaying given brand of lactase:
(two sub teams each perform an assay) 10/25/94, rvsd 18 Sept ’95, 20 Sept. ’96
mortar and pestle
100 mL graduated cylinder
5.0 mL pipet (for dH 2O)
pipet bulb or helper
2 x 200 lambda micropipettes
(for ONPG and enzyme)
2 x 1000 lambda micropipettes
(for buffer and 4% K 2CO3 )
2 16 x 150 mm test tubes
10 13 x 100 mm tubes
two test tube racks for 13×100 tubes
37C hot block for 13 x 100 mL
spectrophotometer, warmed up
cuvettes in rack
100 mL 0.01 M PO 4 buffer
(to suspend and dilute enzyme)
30 mL dH 2O in 125 mL flask
(to make up assay set)
3 mL 20 mM o-nitrophenyl–D galactoside
15 mL 0.1 M PO 4 buffer, pH 7.0
(for assay tubes)
15 mL 4% K 2CO3
CALCULATION OF LACTASE ACTIVITY/TABLET:
If 1 unit of lactase produces an OD of 1.000/15 min., and the assay was run for 15 mins:
units/tablet = A 450 x 100 mL/tablet x dilution factor x 1/(aliquot in mL)