LACTASE ASSAY: REAGENTS

©David B. Fankhauser, Ph.D.,
Professor of Biology and Chemistry
University of Cincinnati Clermont College,
Batavia OH 45103
Suspending lactase
in buffer
This page has been accessed Counter times since 31 October 2002. 
19 September 1993. rvsd 25 October 1994, 18 Sept 95, 20 Sept. '96
[See Hartman, Suskind & Wright, Principles of Genetics Lab Manual, (1965). pp. 52-58.)]
Lactase assay,
after incubation

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

EQUIPMENT:
mortar and pestle
100 mL graduated cylinder
ice bath
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 13x100 tubes
37C hot block for 13 x 100 mL
2 stopwatches
spectrophotometer, warmed up
at spectrophotometer:
cuvettes in rack
wipettes
 

SUPPLIES:
lactase tablets
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
(ONPG)
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)