Question

I need to change the following experiment to measure thefluorescence in not one but 3 different pH levels. I can keep mostof the procedure, but I am not sure what I need to change in orderto measure the correct data.
http://probes.invitrogen.com/media/pis/mp22013.pdf



Experimental Protocol
The following procedures are designed for use with afluorescence
multiwell microplate reader. For use with a standardfluorometer,
volumes must be increased accordingly.
Reagent Preparation
1.1 Prepare a DQ lysozyme substrate stock suspension. Prepare
a 1.0 mg/mL stock suspension of the DQ lysozyme substrate
by suspending the contents of the vial (Component A) in
1.0 mL of deionized water (dH2O). The DQ lysozyme substrate
suspension can be stored at 4°C for at least one month. For
long-term storage (up to six months), add 2 mM sodium azide
and store at 4°C or freeze at -20°C in single-use aliquots.PROTECT
FROM LIGHT. AVOID FREEZE–THAW CYCLES.
1.2 Prepare a 1000 U/mL lysozyme stock solution. Dissolve
the contents of the vial (Component C) in 1 mL of dH2O. Thereconstituted
lysozyme can be frozen in aliquots and stored at -20°C
for at least six months without significant loss of activity.
AVOID FREEZE–THAW CYCLES.
Lysozyme Activity Assay
The following protocol describes the assay of lysozymeactivity
in a total volume of 100 µL per microplate well. The kitprovides
sufficient material for ~400 assays.
2.1 Prepare a lysozyme standard curve. Fill 8 wells with 50µL
of 1X reaction buffer (Component B). Add 50 µL of the 1000U/mL
stock solution of lysozyme (prepared in step 1.2) to the firstwell,
mix by pipetting, then transfer 50 µL to the second well.Repeat
this process from one well to the next, except discard 50 µLfrom
the mixture in the seventh well and add nothing to the eighth
well. Thus, the lysozyme concentration will range 500 U/mL to
0 U/mL in the 50 µL volumes, for a range of 250 U/mL to 0 U/mLin
the final 100 µL volumes.
2.2 Dilute the experimental samples. Dilute thelysozymecontaining,
experimental samples in 1X reaction buffer (Component
B). A volume of 50 µL will be used for each reaction and
the concentration will be twofold lower in the final reactionmixture.
A variable dilution will be required depending on the
lysozyme activity present in the sample. In the first trial,the
samples should be serially diluted to determine the optimal
sample concentration.
2.3 Prepare a DQ lysozyme substrate working suspension.
Prepare a 50 µg/mL working suspension of the DQ lysozyme
substrate by diluting the 1 mg/mL stock suspension (preparedin
step 1.1) 20-fold in 1X reaction buffer (Component B). A 50µL
volume will be used for each reaction. For example, toprepare
enough working solution for 20 assays including the standard
curve, add 50 µL of DQ lysozyme substrate stock suspension to
950 µL of 1X reaction buffer. Note that the final concentrationof
the DQ lysozyme working suspension will be twofold lower in
the final reaction buffer.
2.4 Begin the reaction. Add 50 µL of the 50 µg/mL DQ
lysozyme substrate working suspension (prepared in step 2.3)to
each microplate well containing the experimental or thestandard
curve samples.
2.5 Incubate the reaction mixtures. Incubate the mixtures
for 30 minutes or longer at 37°C, protected from light.Because
the assay is continuous (not terminated), fluorescence can be
measured at multiple time points to follow the kinetics ofthe
reaction.
2.6 Measure fluorescence. Measure the fluorescence intensity
of each reaction in a fluorescence microplate reader equipped
with standard fluorescein filters. Digestion products fromthe
DQ lysozyme substrate have absorption maxima at ~494 nm and
fluorescence emission maxima at ~518 nm.
2.7 Correct for background fluorescence. Subtract the value
derived from the no-enzyme control. Determine the lysozymeactivity
of the experimental samples from the standard curve.