In case you have any further technical questions, please contact our technical support team:

1. What information do I gain from ATPase assays?

   Membrane vesicles containing ABC transporters can be used to measure the ATPase activity of the transporter. Changes in the activity of the transporter in the presence of a test drug indicate that there is an interaction between the test drug and the transporter assayed.

2. What information do I gain from vesicular transport assays?

   Membrane preparations, if they contain inside-out vesicles in adequate amount, can be used to measure the actual transport of the molecules using the vesicular transport assay. If the test drug is a transported substrate of the transporter it will translocate in this assay from the buffer into the membrane vesicles. Membrane vesicles can be separated from the rest of the compounds by rapid filtration and the amount of transported substrate can be measured. In laboratories lacking the proper analytical hardware (LC/MS/MS or HPLC) vesicular transport assays can be run in a competition format using radioactive reference substances.

3. What information do I gain from whole cell-based assays (Calcein assay for MDR1 and MRP1, Hoechst dye extrusion assay for MXR)?

   Whole cell based assays provide information that is relevant to living cells. These methods, together with ATPase and vesicular transport, can give a clear insight into the interaction of the test compound with the transporters assayed. In case of ambiguous results, whole cell assays can be used to confirm data generated on vesicular transport and ATPase assays.

4. Are membrane products supplied with the two assays (ATPase or vesicular transport) the same or different in any way?

   Membrane preparations always contain some closed membrane vesicles that are in inside-out orientation (under 16% of total lipid). In these inside-out vesicle preparations, the transport of substrates across the membrane takes molecules from the buffer into the membrane vesicle. Vesicles sold as "for vesicular assays" are validated for both vesicular and ATPase assays, while vesicles sold as "for ATPase assays" are validated only for ATPase.

5. Does freezing and thawing ruin membrane vesicles?

   Repeated freezing and thawing has very little effect on the ATPase activity of the transporters. Yet, it can ruin the vesicular structure of the membrane preparation. Thus, it is recommended to store membrane preparations in small aliquots.

6. What ATP brand is suitable for ATPase assays?

   The ATPase assay in very sensitive to the quality of the ATP used. ATP is available from several vendors, but unfortunately some batches do not meet the quality requirements necessary to run the ATPase assay. We have very good experience with ATP purchased from Sigma (see protocol for catalogue numbers), so if you are concerned with the quality of ATP we suggest purchasing from this vendor.

7. Why add glutathione to the assay mix in MRP1 and MRP2 membrane based studies?

   The MRP1 and MRP2 transporters are known to transport some small, positively charged or hydrophobic molecules only in the presence of glutathione (co-transport), which is readily available in living cells. Also, in the ATPase assay some drugs only modulate the ATPase activity of the MRP1 and MRP2 transporters in the presence of glutathione. Therefore, we suggest assaying the interaction of the test drugs with the MRP1 and MRP2 transporters with and without the presence of 2 mM glutathione (added to the assay mix).

8. What organic solvents can be used in what concentration without affecting the performance of the assay?

   Membrane-based assays tolerate the most widespread organic solvents used in the pharmaceutical industry (DMSO, ethanol, methanol) up to 2%. Whole cell-based assays are more sensitive; we suggest keeping the concentration of these solvents under 1%.

9. What reference substrates do you use?

   You can find them for each different assay in the downloadable ABC Transporter Membrane and Fee-for-Service Screening Product Catalogue.

10. How many substances can I test with 1 mg membrane preparations in the ATPase assay?

    We perform the ATPase assay on 96-well plates, adding 20-40 µg of membrane preparation per well. We run 8 concentrations, 2 duplicates and two types of assays (activation + inhibition) per compound. This way we normally test 2 compounds per plate, using up about 76 wells (64 assay + 12 control), using ~3 mg membrane (~1.5 mg membrane / compound). For basic screening the number of assays per compound can be decreased according to one's actual needs.

11. How many substances can I test with 1 mg membrane preparations in the vesicular transport assay?

    We recommend to use 50 µg membrane preparations per well for transport inhibition assay readout or HPLC analysis, or LC/MS-MS analysis. Regarding testing strategies, see the ATPase description above.

12. Can I perform the vesicular transport assay also with different readouts e.g. HPLC or LC/MS-MS?

    Our protocol is optimized for a competition type assay set-up, using a labelled reporter compound, however, HPLC or LC/MS-MS can also be used.

13. Why does SOLVO use mostly Sf9 insect cells instead of mammalian cells?

    The baculovirus-insect cell system has many advantages over mammalian cells or the membranes prepared from mammalian cells.

    • The baculovirus insect cell system is easy-to-use and gives high expression of the transduced gene.
    • Obviously no other mammalian transporters are present in the insect cells.
    • In addition, the endogenous expression of the insect cell proteins is down-regulated upon baculovirus infection.
    • The MDR transporter expression profile in some cell-lines, particularly the drug-selected ones is somewhat unstable. Additional care must be taken for standardization, validation on a regular basis.

    One exception is the MXR protein, where membranes derived from mammalian cells are also used. This is a very special case, where Sf9 cells have a major drawback against Sf9 cells in the ATPase assay (for details see FAQ #17)

14. Is it possible to obtain the expression systems (recombinant baculovirus or selected cell lines) from SOLVO Biotechnology for in-house Sf9/mammalian membrane production?

    It is not our business policy to sell recombinant baculoviruses containing the transporter cDNA or the virus-infected Sf9 cells directly, or the selected mammalian cell lines. We provide well-manufactured and standardized membrane products, equipped with datasheets to enable a strict quality control. We believe that a long-term membrane support agreement is more economical for our customers than dedicating their own resources to membrane production. It is our experience that producing standardized, high-expression level membrane preparations has caused problems even in some wellprepared labs at large pharmaceutical companies.

15. Are the membranes patented? Do I need sublicense for cDNA?

    The preparation of Sf9 membrane products includes a step of transfection with cDNA. For some corporate users, cDNA usage sublicences might be required from third parties for some ABC transporters in some countries. Although we truly believe that our customers are not exposed to any risk of patent infringement, this is not a fully clear situation in case of some ABC transporters (mainly in the US, where cDNA is patentable – in the EU one cannot patent cDNA). Membrane products from selected mammalian cell lines are not exposed to such potential legal risk.

16. What is the difference between the wild type and the mutant (R482G) form of the MXR transporter?

    SOLVO Biotechnology offers two variants of the MXR(ABCG2) transporter: the wild type form and one mutant variant. So far only the wild type variant was found in the human population, while the mutant variant was present in some drug-selected cell lines. The substrate specificity of the two forms has also been reported to be different (Ozvegy et al., 2002). Vesicle preparations containing the wild type transporter can be used in human pharmacokinetic studies, while preparations containing the mutant variant are recommended to be utilized in academic research.

17. Which MXR membrane preparation should I use in my ATPase and Vesicular Transport assays: Sf9 or mammalian?

    The MXR transporter shows a significantly higher intristic ATPase activity than most ABC transporters. This basal activity has been attributed to activators (MXR substrates) that are present in the membrane preparations. Depending on the type of membrane preparation, this activating effect can be different. In case of membrane preparations from insect cells (SB-MXR-Sf9-ATPase), the baseline activity is very high, so most interacting compounds actually inhibit this baseline activity. In case of membranes prepared from mammalian cells (SB-MXR-M-ATPase), the baseline activity is lower, and some interacting compounds inhibit, while known transported substrates activate the baseline activity. Therefore, the membrane preparations from mammalian cells can be used to search for transported substrates, while the insect membrane preparation is suitable for screening interacting compounds. Also, membranes from mammalian cell lines have higher endogenous ATPase levels, which result in a higher non-transporter related background in the ATPase assay. In vesicular transport assays, the two types of membranes behave similarly.