Frequently Asked Questions

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  • Can I use the TRANSIL assay kit for compounds that are unstable?

    When a compound is unstable and degrades within hours during equilibrium dialysis results will not be useful. However, TRANSIL assay kits have recommended incubation times of up to 12 minutes (all 96 well kits) or 30 minutes (TRANSIL High Sensitivity Binding Kit) and these incubation time can safely be reduced to only 2 or 10 minutes, respectively. Also, TRANSIL kits are incubated at room temperature, not at 36°C. Most critical compounds remain stable under these conditions.

  • Can I use the TRANSIL HSA Binding Kit for compounds with plasma instability?

    When a compound is unstable in plasma, equilibrium dialysis frequently fails to report adequate results. However, the TRANSIL HSA binding assay performs well under these conditions, because the incubation time is 12 minutes or less at room temperature and there are no enzymes present in the assay system that would degrade the compound.

  • Why is it useful measuring the dissociation constant KD instead of the fraction unbound?

    Healthy humans have a plasma composition that breaks down into 60% albumin and 40% globulins. The latter break down into 4% α1 globulins, of which α1 acid glycoprotein is only a small part, 8% α2 globulins (e.g. haptoglobulin, macrogobulin etc), 12% β globulins, and 16% γ globulins (i.e. our antibodies). However, these are textbook number referring to healthy humans. The plasma composition changes with age, race, sex, and disease condition and the unbound fraction of a drug can change accordingly (Figure 3). In fact, it has been shown that, for example, a 2-fold change in the AGP (AAG) concentration can lead to a 3-fold reduction in survival time of NSCLC patients treated with docetaxel. Thus, measuring the dissociation constant KD of your discovery compound to the major plasma proteins HSA and AGP can be critical in many indication areas.


    Figure 2: Illustration of the reduction of the unbound fraction in response to an increasing AGP (AAG) content of human plasma.
  • Can the TRANSIL High Sensitivity Binding kit be used to assess plasma binding of peptide drugs?

    Yes, the TRANSIL High Sensitivity Kit (or HSB kit) has been successfully used to measure plasma protein binding of peptide drugs, so that there is an easy to use tool for optimizing their half-life. We do provide the kit both in silanized glass vials or PP tubes to assure compatibility with non-specific binding characteristics of your compound. Some peptide drugs with a structure similar to Liraglutide or Semaglutide are better handled in a different assay design. Please inquire to learn more by sending an email to

  • Why are membrane permeability and protein binding important parameters?

    Membrane permeability affects drug absorption and protein binding affects its distribution in the body. Hence, both parameters are key indicators for bioavailability of drug candidates in the developmental pipeline. For effective and efficient drug development pharmacokinetic properties, drugs safety and efficacy need to be optimized at the same time. The TRANSIL products determine the pharmacokinetic parameters and distinguish potential safety problems early on in the drug discovery process.

    Membrane affinity describes a small molecules’ ability to permeate through lipid membranes. Therefore it is directly related to the permeation rate across membranes such as the intestinal membrane or the blood brain barrier.

    Total intestinal absorption depends not only on permeability across the intestinal epithelium, but also on the drug’s pH dependent solubility in the intestine and the passage time.

    Equilibrium distribution of drugs between blood and brain depend not only on the permeation across the brain’s endothelial membranes but also on the extent of a drug’s plasma protein binding.

  • What is the typical assay set-up and protocol?

    One 96-well plate based TRANSIL assay kit can be used for 12 compounds, utilizing 8 wells of one column for one compound. Two wells are used as references to account for potential non-specific binding. Six wells contain TRANSIL beads in increasing concentration. Thus, there are not only six replicates of each measurement; these replicates also vary systematically in binding, such that several internal quality control measures can be applied to validate the assay results.

    Assay Protocol
    1. Drug candidate preparation: prepare 16x stock solution
    2. Drug candidate addition: add 15 µl of the 16x stock solution to each well
    3. Mixing: two variants
      • (A) Mix and resuspend beads by aspirating and dispensing (20x) a volume of 120 µl
      • (B) Incubate the plate on a 3D shaker (e.g. Eppendorf Mixmate) at 800 rpm for 12 minutes
    4. Centrifugation and supernatant sampling:
    • Spin the plate in a swing-out centrifuge for 10 minutes at 750 g
    • Transfer 50 to 100 µl supernatant to a standard 96 well plate for quantification either by UV, HPLC or LC/MS/MS
  • What is the principle of TRANSIL assay kits?

    TRANSIL assays can be regarded as a form of smart dialysis. Instead of establishing a binding equilibrium across a small dialysis membrane, proteins or lipid bilayers expose their surface on immobilized beads directly to the test items. In fact, the surface area of these beads almost the size of a football field making the interactions very fast, so that the binding equilibrium is attained very quickly.


    Another shared feature of all TRANSIL assays is the incubation of a constant concentration of the test compound six different concentrations of beads and two reference wells without any beads. That means, the same drug concentration is exposed to six different protein concentrations or membrane surface areas. The beads are removed by centrifugation, once the compounds have reached a binding equilibrium to the assay’s matrix. The user than samples the supernatants to determine the remaining test compound concentration. The data are entered into a provided spreadsheet and the binding parameters are calculated automatically. The concentration of the test compounds depends on the detection system applied by the user. Typical concentrations are 0.5 to 2µM.

  • Is it possible to use organic solvents in TRANSIL assayS?

    TRANSIL beads are not suitable for use in pure organic solvents or in detergent solutions. However, solubility mediators like DMSO cause no bleeding of the lipids or immobilized proteins when used at low concentrations of up to 2% or even 5%. Consider that organic solvents may form a third phase within the assay system and can thus bias the results. Thus, we recommend a DMSO concentration of 1%.

  • In what kind of format are the TRANSIL products available?

    The standard format of our assay kits are ultra-low binding 96-well plates. These plates have removable wells covered with capbands. This allows users to use each column of a plate individually, so that there is no need to run 12 compounds at the same time.

    Upon request, we are able to produce all kits in custom formats, like low binding Eppendorf caps or silanized glass vials. Please contact us if you require customized formats such as 384-well plates.

  • How are the proteins orientated on the bead surface?

    The proteins are randomly orientated on the bead surface. This way, all binding sites are exposed and no loss of function due to the covalent immobilization procedure is observed.

  • How many measurements are possible with the TRANSIL kits?

    The 96-well format allows the determination of 12 pharmaceutical drug candidates, using 8 wells per drug candidate. Muliplexing can be used safely in the membrane binding kits (see Longhi et al. (2011) DMD 39(2)312-321.

  • What is the buffer system of the TRANSIL kits?

    TRANSIL beads are suspended in PBS buffer (10 mM phosphate buffer with 155 mM NaCl, pH 7.4).

  • Why is plasma protein binding important?

    The extent of binding to plasma influences the way in which a drug distributes into tissues in the body. If a compound is highly bound, then it is retained in the plasma, which results in a low volume of distribution. This may impact on the therapeutic effects of the compound by limiting the amount of free compound which is available to act at the target molecule. Extensive plasma protein binding also limits the amount of free compound available to be metabolized which can, in turn, reduce the clearance of the compound.

  • How does the TRANSIL intestinal absorption compare to octanol/water partitioning?

    Frequently membrane permeability is approximated by octanol/water partitioning. The drawback of this system is that permeation of small molecules through membranes depends on structural interactions between the test substance and the membrane such as hydrogen bonding Van der Waals interactions. Since octanol is structurally very different from phospholipid bilayers, it cannot model these interactions well. Octanal/water partitioning has been shown to be particularly poor in predicting permeability of ionized compounds. Our membrane beads are just as good at predicting permeability of ionized and non-ionized compounds and compare well to permeation into liposomes, which is regarded as the gold standard in permeability studies. Moreover, with an incubation of only two minutes our technology is more easily scalable to higher throughput than octanol/water or liposome/water partitioning.

  • How do TRANSIL membrane permeability assays compare to PAMPA?

    PAMPA assays use various unstructured lipid mixtures on filter membranes. Since the lipids do not have the structure of true phospholipid bilayers, they cannot mimic the structure and charge interactions between the test substances and the membranes nearly as well as the TRANSIL assay kits.

    Also, non-specific binding confounds PAMPA measurements more strongly than TRANSIL assays. Filter membranes are well known to attract sticky compounds. This interaction with the filter membranes results in biased or even failed permeability estimates.

    PAMPA assays require overnight incubation while TRANSIL plates require only 2 minutes incubation time.

    PAMPA plates have to be prepared and validated in your laboratory, while TRANSIL plates are ready to use and validated by us.

  • How stable are TRANSIL beads?

    Stored at -20 °C the TRANSIL beads have a shelf life of 2 years and we guarantee their stability for at least 6 months after delivery.

  • What are dissociation constants of marketed drugs?

    The table below lists dissociation constants for some marketed drugs.

    Compound KD(HSA) in μM KD(AGP) in μM
    Propranolol 430.0 5.2
    Imatinib 51.8 1.3
    Vincristine 123.0 3.9
    Paclitaxel 62.0 58.6
    Docetaxel 15.0 3.3
    Verapamil 357.0 6.1
    Sulfasalazine 0.6 27.0
    Ketoprofen 1.7 289.0
    Warfarin 6.4 8.8
    Chlorpromazine 38.0 77.6
    Progesterone 8.5 10.3
  • Why can results from the TRANSIL HSA Binding kit differ from literature values?

    Most literature values were obtained by dialysis experiments. Usually, dialysis experiments use pooled plasma that is not controlled for its exact protein composition. However, even minor changes in both HSA and AGP concentration can have major effects on the fraction unbound of some drug candidates. Also, most literature values date back to times when there was no awareness of the influence of pH shifts during the dialysis experiment on the results. And only recently has it been recognized that plasticizers in plasma collection bags can have an equally dramatic effect on plasma protein folding and thus binding of drugs. Thus, differences between results from TRANSIL and dialysis experiments are to be expected due to artefacts that can arise and uncontrolled variables in dialysis.

  • Can TRANSIL assay kits be handled by pipetting robots?

    The TRANSIL assay kits can be easily used in an automated environment. The assay plates are compliant to SBS (Society of Bimolecular Screening) standards and are already filled with fixed volumes of buffer (PBS) and TRANSIL beads. They ship them frozen on cool packs and they come ready-to-use. The assay starts with the test item addition in one pipetting step. After mixing and incubation for 2 minutes, the beads are separated by low speed centrifugation (5 minutes with 750 g). A fixed volume of the supernatant or is transferred into a new plate for quantification by UV, HPLC, or LC/MS.

  • What are the key liquid handling issues for automation?

    An important point is the set up of the mixing step to ensure a complete re-suspension in the well, including the exact programming of the mixing velocities to avoid cross contamination. After separation by centrifugation the immersion depth plays an important role to avoid displacement of the beads. For the transfer of the supernatant the needles of the pipetting device should be placed at least 2 mm above the settled beads within the wells. Usually that is achieved by placing the needles at least 11 mm above the zmax-position within the wells, which is defined as the maximum immersion depth.

  • What is the final concentration of the compound in the assay?

    We recommend that compounds are used at final concentrations that range between 0.5 and 70 μM per well if possible. A compound concentration of 50 μM is a good starting point for UV detection, while for LC/MS/MS we recommend using 0.5 to 2 µM concentrations. Higher concentrations may cause interference and biased binding or permeability estimates.

  • Are TRANSIL beads also available in other buffer systems?

    Generally all assays are performed in PBS at the pH of 7.4. Please contact us if you require different buffer systems.

  • Is it possible use TRANSIL beads in different pH environments

    Absorption of an orally administered drug is closely linked to the pH value of the monitored compartment. The gastrointestinal fluid’s pH differs vastly along the passage through the gastrointestinal tract. Hence, total absorption and thus bioavailability depends upon permeability rates of the full range of ionization stage of a drug. TRANSIL Intestinal Absorption kits allow the determination of membrane permeability in a range from pH 3 to 11. Thus, the TRANSIL technology allows estimation of membrane permeability across the full physiologically relevant pH range.

  • What is the TRANSIL technology?

    The core of the TRANSIL technology are silica beads with specific surface chemistry. The beads are modified such that they covalently bind proteins or non-covalently bind membranes to their surface. Protein covered beads have very little exposed bead surface. And the small fraction of exposed surface area is further modified to minimize unspecific binding of test substances. Membrane beads carry true phospholipid bilayers of natural membrane composition. These membranes are reconstituted in our laboratory from natural materials and mimic precisely true membrane properties such as fluidity and molecular interactions.

    The key advantage of the TRANSIL technology is that the products are all based on natural materials that are reconstituted in a highly defined way in our laboratories, such that our assays offer high reproducibility. At the same time they measure the key parameters of interest directly, since they are based on the natural cell components.

    The beads in one 96-well plate have roughly the surface area of a tennis court. This allows very rapid detection of protein binding or membrane permeability. Hence, our assay kits require only two minute incubation time (Note: to avoid any issues arising from unexpectedly slow on and off rates we recommend an incubation time of 12 minutes in our user guides).

  • What is TRANSIL for Protein Binding?

    The TRANSIL Binding kits contain beads covered with a specific plasma protein allowing the determination of the bound and free fraction of a pharmaceutical drug. The bead surfaces are designed such as to minimize interactions with drug molecules. This prevents non-specific binding. We offer three different types of proteins that are linked to the surface of the beads: Human serum albumin (HSA), alpha-1 acidoglyoprotein (AGP), rat serum albumin (RSA), mouse serum albumin (MSA) and upon request bovine serum albumin (BSA).

  • Which kind of UV plates should be used for UV read-out?

    For 96 well plates we recommend the Corning 96 well half area plates (No. 3679). Please check that the background absorbance above 250 nm is negligible for quantification.

  • What are the prerequisites to use quantification by UV?

    Quantification by UV is only possible for compounds with significant absorbance above 250 nm due to the background absorbance of most commercially available translucent plates and DMSO. We recommend centrifugation of UV plates for 2 minutes at 750 g to destroy any air bubbles that otherwise may cause light scattering.