4 Notes
1. The 20Â Reaction buffer provided in the EnzChek Kit contains
2 mM of sodium azide, that acts as a preservative. The Kit
allows to perform the activity assay over a pH range of
6.5–8.5, but in this case the buffer must be prepared with the
pH of interest (here, sodium azide can be omitted). To generate a pH curve, we recommend to prepare a 20Â stock of
Reaction buffer at pH 9.5. Then, distribute it in 5 mL aliquots
(as many as pH values to be tested). Adjust the pH of every
aliquot to the desired value with 37% HCl and then add milli-Q
water up to 10 mL to get 10Â aliquots of each pH. Remember
that the volume of these 10Â aliquots added per activity reaction must be doubled (as the recipe in Table 1 contains a 20Â
Reaction buffer).
2. The Kit includes 6.3 mg (20 μmol) of MESG. Add 20 mL of
milli-Q water directly to the container to prepare a 1 mM stock.
Immediately, aliquot the homogenized MESG solution and
store the aliquots at À20
C. As each individual reaction
requires 40 μL of MESG substrate, and normally triplicates
will be performed per condition, we recommend to prepare
both 200 μL and 500 μL aliquots. Immediately before use,
thaw the required aliquot of MESG at 37
C (no more than
5 min), vortex strongly and place on ice. If more than one
aliquot is needed, we recommend to mix all of them together
and use the same solution for all reactions. As described in the
manufacturer’s instructions, MESG solution is stable on ice at
least for 4 h at pH 7.5. Be aware that the half-life of this
substrate may change in different conditions. It is not recommended to freeze and reuse MESG leftovers.
3. The Kit provides two vials of 50 U of freeze-dried PNP. To
prepare a 100 U/mL stock solution add 0.5 mL of milli-Q
water to one of the vials. This solution can be stored at 4
C for
at least 1 month. The nonreconstituted PNP vial may be stored
at À20
C.
4. The Kit contains one vial of 6 U of lyophilized inorganic
pyrophosphatase. Add 200 μL of milli-Q water to the vial to
prepare a stock of 30 U/mL and store it at 4
C. Before
performing the activity assay, prepare a 3 U/mL aliquot by
diluting tenfold the 30 U/mL stock into 1Â Reaction buffer
(previously diluted from 20Â Reaction buffer). Never dilute
the inorganic pyrophosphatase into 20Â reaction buffer. Leftover 3 U/mL solution can be stored at 4
C for 1 week.
5. The Kit provides 500 μL of 50 mM Na 4 P 2 O 7 (with 2 mM
sodium azide) as a source of PPi. Prepare a 500 μM working
solution of PPi standard by diluting 100-fold a portion of the
Determination of GGPP Synthase Activity
33
1. The 20Â Reaction buffer provided in the EnzChek Kit contains
2 mM of sodium azide, that acts as a preservative. The Kit
allows to perform the activity assay over a pH range of
6.5–8.5, but in this case the buffer must be prepared with the
pH of interest (here, sodium azide can be omitted). To generate a pH curve, we recommend to prepare a 20Â stock of
Reaction buffer at pH 9.5. Then, distribute it in 5 mL aliquots
(as many as pH values to be tested). Adjust the pH of every
aliquot to the desired value with 37% HCl and then add milli-Q
water up to 10 mL to get 10Â aliquots of each pH. Remember
that the volume of these 10Â aliquots added per activity reaction must be doubled (as the recipe in Table 1 contains a 20Â
Reaction buffer).
2. The Kit includes 6.3 mg (20 μmol) of MESG. Add 20 mL of
milli-Q water directly to the container to prepare a 1 mM stock.
Immediately, aliquot the homogenized MESG solution and
store the aliquots at À20
C. As each individual reaction
requires 40 μL of MESG substrate, and normally triplicates
will be performed per condition, we recommend to prepare
both 200 μL and 500 μL aliquots. Immediately before use,
thaw the required aliquot of MESG at 37
C (no more than
5 min), vortex strongly and place on ice. If more than one
aliquot is needed, we recommend to mix all of them together
and use the same solution for all reactions. As described in the
manufacturer’s instructions, MESG solution is stable on ice at
least for 4 h at pH 7.5. Be aware that the half-life of this
substrate may change in different conditions. It is not recommended to freeze and reuse MESG leftovers.
3. The Kit provides two vials of 50 U of freeze-dried PNP. To
prepare a 100 U/mL stock solution add 0.5 mL of milli-Q
water to one of the vials. This solution can be stored at 4
C for
at least 1 month. The nonreconstituted PNP vial may be stored
at À20
C.
4. The Kit contains one vial of 6 U of lyophilized inorganic
pyrophosphatase. Add 200 μL of milli-Q water to the vial to
prepare a stock of 30 U/mL and store it at 4
C. Before
performing the activity assay, prepare a 3 U/mL aliquot by
diluting tenfold the 30 U/mL stock into 1Â Reaction buffer
(previously diluted from 20Â Reaction buffer). Never dilute
the inorganic pyrophosphatase into 20Â reaction buffer. Leftover 3 U/mL solution can be stored at 4
C for 1 week.
5. The Kit provides 500 μL of 50 mM Na 4 P 2 O 7 (with 2 mM
sodium azide) as a source of PPi. Prepare a 500 μM working
solution of PPi standard by diluting 100-fold a portion of the
Determination of GGPP Synthase Activity
33