4. Prepare test compounds
.
a.Spectroscopic purity DMSO for test compound
preparation.
b.Add DMSO to test compounds according to the table
below:
c.Add 80 µl of each test compound solution into each
well of the StockPlate.
d.Extra test compound can be frozen for future use.
5. Populate the worksurface with appropriate labwares.
a.Place 3 96-well UV plates on worksurface position.
b.Place Biomek P200 tip boxes on worksurface position.
c.Place PAMPA Sandwich Plate (pre-loaded) on
worksurface position.
d.Place SUPPORT Plate on worksurface position.
e.Place the StockPlate on worksurface position.
f.Place the LipidPlate on worksurface position.
g.Place ASB solution Low Profile Reservoir on
worksurface position.
h.Place System Buffer High Profile 12 Trough Reservoir
on worksurface position.
i.Place Deep well plate for diluted test compounds on
worksurface position.
6. Enter test compound information into Excel template file.
a.Launch Microsoft Excel application.
b.Select
File | Open.
c.Browse to open
PAMPA Template.xlt
file located in
the PAMPA Evolution command Software directory.
d.Enter each compound in the same well location that it
was loaded in the StockPlate.
e.Physiochemical constants are optional.
f.Do not leave blank spaces in the Excel template sheet.
g.Rename the Excel file and save in DATA sub-folder in
the PAMPA Evolution command Software.
h.Exit the Excel application.
7. Run PAMPA assay.
a.Click the
Import from Excel
button on the toolbar.
b.The Input Compounds dialog box appears, click the
Browse
button to import test compound Excel
template file.
c.Click the
OK
button for the
Warning
dialog box for
correct StockPlate type.
d.Verify the loaded compound names and
physiochemical constants, click the
OK
button
to
continue.
e.Open the
Instrument-Settings
, select the
Use pre-
measured blank
.
f.Browse to select
DefaultBlankPlate.spc
.
g.Uncheck
Tell me about low-UV wells…
h.Click the
OK
button
and exit the
Instrument Settings
.
i.Click the
General Information
button, fill in the
appropriate experimental conditions, click the
OK
button.
j.Click the
Start Assay
button.
k.Select the
Uninterrupted Assay Flow
and
Use pre-
measured blank
check boxes.
l.Verify that
Double-Sink Protocol
is selected, and
incubation time is set to “0.5 hr”.
m.Select
Properties
and verify the System Solution
Volume = 600 µL and Stirring be ABL = 40 µM.
n.Select
Multi pH Assay
and select
pH Map
.
o.Verify the updated layout of System Solution at
different pH.
p.Click the
OK
button and
Start
the assay to proceed.
Test Compounds
MW
Weight (mg)
DMSO (ml)
Concentration (mM)
Ketoprofen
254.3
2.54
1
10
Verapamil
491.1
4.91
1
10
Antipyrine
188.2
9.40
1
50
Metoprolol
684.8
6.85
1
10
Carbamazepine
236.3
2.36
1
10
Ranitidine
350.9
3.51
1
10
Propanolol
295.8
2.96
1
10
DMSO
Ketoprofen
Verapamil
Antipyrine
Metoprolol
Carbamazepine
Rantidine
Propranolol