module (seeNote 4). A security guard cartridge pre-column
(SUPELCOSIL™LC-18-DB Supelguard™Cartridge, 5μm,
2cm 3 mm, Sigma-Aldrich 59565C30 SUPELCO) is
advised (but not mandatory) for protecting the LC-18 column.- Set the column compartment temperature at 30C.
- Purge the system.
- Start the pump with a gradual increase in pyrophosphate buffer
flow rate until 0.8 ml/min for ~1 h. The pressure should be
continuously monitored and do not exceed the capacity of the
system (usually<100 bar, but this could vary depending of the
HPLC tubing used). - Reduce the flow rate to ~0.1 ml/min and let the system equili-
brate overnight.
3.4 Preparation
of the Samples
- Defrost the standard stocks and samples on ice.
- Vortex briefly and spin down the samples at 8000gfor 2 min
at 4C. - Prepare a standard solution containing ATP, ADP, and AMP at
a final concentration of 30μM by mixing 45μl of each standard
in 1365μl of pyrophosphate buffer. - Transfer 250μl of the standard solution in triplicate in a
96-well plate. Keep the plate on ice. - Prepare each sample on ice in the 96-well plate by mixing
100 μl (for tissue lysate) or 150μl (for cell lysate) of sample
extract with 150 μl or 100 μl of pyrophosphate buffer,
respectively. - Seal the 96-well plate with an ad hoc plastic film cover.
3.5 HPLC Run 1. Purge the system.
- Increase gradually the flow rate to 0.8 ml/min and let the
system equilibrate for ~30 min. - Switch on the UV light.
- Transfer the 96-well plate in the temperature-controlled auto-
sampler previously set at 4C. - Program the HPLC software: injection of 20μl of standard or
samples every 20 min. - Start the run (seeNote 5).
- Once the whole run is completed, reduce the pump flow rate. If
multiple runs are planned, go back tostep 2once you are ready
to load the next 96-well plate. Stop the pump immediately (set
flow rate to 0 ml/min), if you want to store the column
(continue withstep 8). In case of unsatisfactory results, such
234 Noemı ́Garcı ́a-Tardo ́n and Bruno Guigas