Calculate the volumes of needed to make solutions of alkaline phosphatase and p-nitrophenylphosphate with the given concentration and update the table below. Show one (1) sample calculation.

Reagents:

Reaction buffer: 0.5 M Tris HCl (pH 8.0) with 5 mM MgCl₂

Alkaline phosphatase: 500 Âµg/mL stock solution in reaction buffer.

p-nitrophenylphosphate: 1000 ÂµM and 10,000 ÂµM stock solution in reaction buffer.

B. Substrate solutions

#

[Substrate] ÂµM

p-nitrophenylphosphate (ÂµL)

Enzyme

((ÂµL)

Buffer (ÂµL)

Total volume (ÂµL)

1

10

100

1000

2

25

100

1000

3

50

100

1000

4

100

100

1000

5

200

100

1000

6

500

100

1000

7

1000

100

1000

8

2000

100

1000

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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 4Â°C for at least one month. For
long-term storage (up to six months), add 2 mM sodium azide
and store at 4Â°C or freeze at -20Â°C in single-use aliquots.PROTECT
FROM LIGHT. AVOID FREEZEâTHAW 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 -20Â°C
for at least six months without significant loss of activity.
AVOID FREEZEâTHAW CYCLES.
Lysozyme Activity Assay
The following protocol describes the assay of lysozymeactivity
in a total volume of 100 ÂµL per microplate well. The kitprovides
sufficient material for ~400 assays.
2.1 Prepare a lysozyme standard curve. Fill 8 wells with 50ÂµL
of 1X reaction buffer (Component B). Add 50 ÂµL of the 1000U/mL
stock solution of lysozyme (prepared in step 1.2) to the firstwell,
mix by pipetting, then transfer 50 ÂµL to the second well.Repeat
this process from one well to the next, except discard 50 ÂµLfrom
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 ÂµL volumes, for a range of 250 U/mL to 0 U/mLin
the final 100 ÂµL volumes.
2.2 Dilute the experimental samples. Dilute thelysozymecontaining,
experimental samples in 1X reaction buffer (Component
B). A volume of 50 ÂµL 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 Âµg/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 50ÂµL
volume will be used for each reaction. For example, toprepare
enough working solution for 20 assays including the standard
curve, add 50 ÂµL of DQ lysozyme substrate stock suspension to
950 ÂµL 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 ÂµL of the 50 Âµg/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 37Â°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.

Information

Day 1. Synthesis and characterization of HA and CHA. Preparation of ceramics. Prepare two solutions, 100 mL each: (1) 0.5 M Ca2+ solution (from Ca(NO3)2Â·4H2O) with pH adjusted to 11 using 1 M NaOH; (2) 0.3 M solution of PO4 3- ions (from Na2HPO4). For the synthesis of CHA add the carbonate source (NaHCO3) to the phosphate solution such that the PO4 3- : CO3 2- ratio is 5:1 or 2:1. Dropwise add the phosphate and carbonate solutions over the course of 10 minutes to the calcium solution. Heat and mix the solution to 80 Â°C for 1 hour adjusting the pH to 11 if needed by adding 1 M NaOH. Cool it down and vacuum filter your precipitate, wash three times with DI water. Dry the powder in the oven and calculate your yield. Use some of the dry powder to press a pellet. This pellet will be annealed at ~1000 Â°C overnight to fabricate dense ceramic material, which will be used later in the SBF test. Submit some of the sample to your TA for XRD measurements. If time permits, perform FTIR measurements of your sample and compare it to bovine CHA.

Day 2. Preparation of SBF.6 Thoroughly clean 1000 mL glass beaker, try to avoid heavily scratched glassware. 1) Add 960 mL of deionized water and dissolve 6.5456 g of NaCl, stir vigorously at RT for 5 min 2) Dissolve 2.2682 g of NaHCO3 and stir vigorously at RT for 5 min 3) Dissolve 0.373 g of KCl and stir vigorously at RT for 5 min 4) Dissolve 0.1419 g of Na2HPO4 and stir vigorously at RT for 5 min 5) Heat the solution to 36.5-37 Â°C 6) Dissolve 0.3049 g of MgCl2Â·6H2O and stir vigorously for 5 min 7) Add 9 mL of 1 M HCl solution (use a pipette and add it slowly) and stir vigorously for 5 min 8) In small portions add 0.3675 g of CaCl2Â·2H2O and stir vigorously for 5 min 9) Add 0.071 g of Na2SO4 and stir vigorously for 5 min 10) In small portions add 6.057 g of Tris [=(CH2OH)3CNH2]. Upon adding Tris, the solution should become turbid; keep stirring. 11) Insert the pH electrode into the solution, which should be at around 37Â°C until now 12) Add 30 mL of 1 M HCl slowly in 5 mL portions, by using a pipette, your addition plan is 5 + 5 + 5 + 5 + 5 + 5 mL = 30 mL (with the addition of each 5 mL portion of 1 M HCl, pH will gradually drop towards 7.4) 13) Keep stirring and be careful when adding the last â5 mL portionâ of 1 M HCl, add this 5 mL portion quite slowly (i.e., drop by drop) while watching the pH meter reading 14) You can easily adjust the pH at 7.4 (at 36.5 or 37Â°C), pH=7.38, 7.37, 7.41 or 7.39 are also OK 15) Measure the total volume of the transparent solution, if it is not exactly equal to 1000 mL, you must add deionized water to complete the volume to 1000 mL Table 2. Order and amounts of reagents for 1000 mL of SBF.

# Reagent Amount 1 NaCl 6.5456 g 2 NaHCO3 2.2682 g 3 KCl 0.373 g 4 Na2HPO4 0.1419 g 5 MgCl2Â·6H2O 0.3049 g 6 1.0M HCl 9 mL 7 CaCl2Â·2H2O 0.3675 g 8 Na2SO4 0.071 g 9 Tris 6.057 g 10 1.0M HCl 30 mL

Prepare a water bath heated to ~37 Â°C. Put the SBF solution into a 25 mL glass vial and heat it to 36.5 Â°C on a water bath. Place a pellet of ceramic material such that the entire sample is submerged in the SBF, seal the container and leave it at 36.5 Â°C until the next lab period.

question: Not all of the carbonate ions used in the reaction actually incorporate in the final structure of CHA. Propose a characterization method to quantitatively study amount of carbonate in your CHA samples (for instance, carbonate to phosphate ratio, which might be different from what you have in your solution at the beginning). Propose a method to estimate the ratio between A- and B- type carbonates and explain how/why this method can distinguish the A- and B- type carbonate substitution.

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#	[Substrate] ÂµM	p-nitrophenylphosphate (ÂµL)	Enzyme ((ÂµL)	Buffer (ÂµL)	Total volume (ÂµL)
1	10		100		1000
2	25		100		1000
3	50		100		1000
4	100		100		1000
5	200		100		1000
6	500		100		1000
7	1000		100		1000
8	2000		100		1000