PREDEASY Helpdesk

Thank you for purchasing SOLVO PREDEASY™ ATPase Assay Kit product. The PREDEASY™ ATPase Kits include all reagents and solutions needed for the execution of both activation and inhibition assays. It is important to point out that the followings are required to run the assay successfully:

1. General laboratory practice (e.g. pipetting small volumes, 1 µls, etc. )

2. Product performance according to the data sheet specifications is only guaranteed if the assay steps are closely followed and recommended equipment and materials are used.

3. Recommended equipment and materials:

  • Water bath / heating block / thermoshaker (BIOSAN PST-60HL or equivalent), 37 oC (Do not use CO2 incubator because of insufficient heat transfer!)
  • Automatic pipettes, multichannel-pipette with corresponding tips (Eppendorf Research, Multipette Plus or equivalent, Eppendorf pipette tips or equivalent quality tips). Please note that the membrane suspension and solutions are provided in excess quantity, still it might not be sufficient for electronic pipettes.
  • 96 well microtiter plates (COSTAR 3585 or equivalent)
  • Microplate reader with absorbance filter suitable for measuring OD between 590 and 630 nm
  • 2 ml, 5ml, 10 ml tubes
  • MilliQ water (phosphate free water)
  • Dimethyl sulfoxide (DMSO, A.C.S reagent spectrophotometric grade, >=99,9%, Sigma 154938)
4. The membrane preparation is the most sensitive ingredient of the PREDEASY™ ATPase Kit. In case of 3 or 10 plate size kits there are 3 or 10 membrane preparation vials in the box. Do not thaw those vials that you do not intend to use in an hour!

5. If most of the 10 assays provided in a 10-plate size kit are done on separate days, it is recommended to aliquot the Assay mix (Vial B), the Developer (Vial G) and the Blocker (Vial H) after the first thawing, and keep the unused aliquots at -80oC until the day of usage. These ingredients might be sensitive to be thawed and frozen several times.

6. PREDEASY ATPase Kits include internal positive and negative controls. Controls which confirm that the transporter can be activated by a known substrate and the activated transporter can be inhibited by a known inhibitor are included as standard components of every protocol.
  • Internal positive controls (CTRL3, CTRL5) in each assay are designed to validate the assay at one concentration point of the activator and inhibitor for each experiment.
  • Vanadate control points serve as internal negative controls at each test drug concentration. Correction with vanadate control values helps to exclude any non-transporter specific vanadate insensitive effect.


PREDEASY-CTRL Assay Kits are additional controls. Negative control experiments applying control membranes are not used in any computations to determine transporter specific EC50, IC50 or maximal efficacy results. They provide quality type information, i.e. information about the origin of the effects. They can confirm positive answers in case of activation assays or they can help to exclude false positives.


Available PREDEASY negative control kits:

ABC TRANSPORTER PREDEASY ATPase Kit Relevant PREDEASY Negative Control Kit
MDR1-PREDEASY-ATPase Kit Assay ProtocoldefPgp-PREDEASY-CTRL Kit Assay Protocol
ratMdr1b-PREDEASY-ATPase Kit Assay Protocolbetagal-PREDEASY-CTRL Kit Assay Protocol
MRP1-PREDEASY-ATPase Kit Assay ProtocoldefMRP-PREDEASY-CTRL Kit Assay Protocol for MRP1
MRP2-PREDEASY-ATPase Kit Assay ProtocoldefMRP-PREDEASY-CTRL Kit Assay Protocol
MRP3-PREDEASY-ATPase Kit Assay ProtocoldefMRP-PREDEASY-CTRL Kit Assay Protocol
BCRP-HAM-PREDEASY-ATPase Kit Assay ProtocoldefBCRP-HAM-PREDEASY-CTRL Kit Assay Protocol
mouseBsep-HAM-PREDEASY-ATPase Kit Assay Protocolbetagal-PREDEASY-CTRL Kit Assay Protocol

(Please note that the assay protocol provided with the negative control kits support the above listed combination of ABC-PREDEASY-ATPase and PREDEASY-CTRL Kits.)


7. Suggested assay plate map for an activation and inhibition study. (Please note, that test article concentrations in the plate map are final concentrations!)

Plate Map (for two parallels) Controls test drug (µM) test drug (µM) + vanadate test drug (µM) + activator test drug (µM) + activator + vanadate

1 2 3 4 5 6 7 8 9 10 11 12
A Blank 300.00 300.00 300.00 300.00 300.00 300.00 300.00 300.00
B 4 nmol Pi 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00
C 8 nmol Pi 33.33 33.33 33.33 33.33 33.33 33.33 33.33 33.33
D Ctrl1 11.11 11.11 11.11 11.11 11.11 11.11 11.11 11.11
E Ctrl2 3.70 3.70 3.70 3.70 3.70 3.70 3.70 3.70
F Ctrl3 1.23 1.23 1.23 1.23 1.23 1.23 1.23 1.23
G Ctrl4 0.41 0.41 0.41 0.41 0.41 0.41 0.41 0.41
H Ctrl5 0.14 0.14 0.14 0.14 0.14 0.14 0.14 0.14

Blank Assay mix + 10 µl distilled water
4 nmol Pi Assay mix + 10 µl 0,4 mM Pi
8 nmol Pi Assay mix + 10 µl 0,8 mM Pi
Ctrl1 Membrane suspension + 1µl solvent
Ctrl2 Membrane suspension + vanadate (1,2 mM) + 1µl solvent
Ctrl3 Membrane suspension + activator + 1µl solvent
Ctrl4 Activated membrane suspension + vanadate (1,2 mM) + 1µl solvent
Ctrl5 Activated membrane suspension + 1µl inhibitor

Membrane suspension + 1µl test drug form drug plate

Membrane suspension + vanadate + 1µl test drug form drug plate

Activated membrane suspension + 1µl test drug form drug plate

Activated membrane suspension + vanadate + 1µl test drug form drug plate

Video shots of the major and the most critical assay steps of a PREDEASY™ ATPase

Detailed assay steps and videos (For registered visitors only)

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This page contains information about following topics: BBB penetration, Transporter CNS, Intestinal Drug Absorption, Transporter Pharmacokinetics, Transporter Pharmacodynamics, Transporter Reabsorption.