TRANSIL PPB Binding Kit
TRANSIL PPB Binding Kit
The TRANSIL PPB Binding Kit estimates the binding of drugs to human serum albumin (HSA) and human alpha-1 glycoprotein (AGP/AAG) at a physiological ratio to predict plasma protein binding. Internal quality controls provide easy assessment of recovery, experiment and data quality.
The kit consists of ready-to-use 96 well microtiter plates. One plate can be used for measuring HSA binding of up to 12 compounds. The assay requires only 5 steps: (i) addition of drug candidate, (ii) mixing and incubation for 12 minutes, (iii) removal of beads by centrifugation, (iv) sampling of supernatant, and (v) quantification of drug candidate.
The TRANSIL Quality Index (TQI) is based on independent measures derived from the data analysis.
- Overall fit of the data to the standard protein binding model
- Recovery: does the model derived compound concentration equal the true concentration?
- Data consistency: does membrane binding increase proportionally with the increasing TRANSIL bead content in each well?
- Data consistency: are the estimated reference concentrations in alignment with the compound concentration used?
- Missing data and outliers.
One assay plate can be used for 12 compounds. Thus, you’ll need to pipette 15 µl of test item to each of 8 wells and repeat this for all 12 compounds. This takes less than 10 minutes even with manual pipetting. After compound addition, the plate is ready for incubation. You can do this with an electronic 8 or 12 channel pipette by aspirating and dispensing a volume of 120 µl for 15 times. That takes just over a minute for each column or row. In total, that makes 8 to 15 minutes depending on your pipette. When using a pipetting robot with a 96 well head this time decreases to 2 minutes. After incubation, the plate needs to be spun in a plate centrifuge for 10 minutes. The supernatants are then ready for quantification by LC/MS/MS, UV, fluorescence or any other method of your choice.
Thus, the total time the start and end of the experiment varies between 7 and 25 minutes depending upon your equipment.
One assay plate can be used for 12 compounds. A special feature of the 96 well plates used for these kits is that each of the 12 columns can be separated from the plate. Thus, it is possible to use the plate for one compound at a time.
The TRANSIL assay kits utilize Micronic 96 well plates with ultra-low-binding tubes. Standard polypropylene tubes have 41x higher non-specific binding and low-binding tubes from other vendors have 2.6x higher non-specific binding.