The assessment of activities of compounds at the protein level can be done by using biochemical assays as a tool for fundamental drug discovery. Proteins have an intrinsic three-dimensional shape, which is based on hydrophilic/phobic, amino acid exchange, and throughout polarity. This enables the creation of complex surface structures. Most of these structures can form selective binding pockets that are vital for interactions among protein, cell surface receptor binding, ATP binding, DNA binding, and several other substrates. To identify and design small molecules that are able to inhibit and activate protein target functionality, it is essential to understand the working of binding pockets. The use of characterized substrate and tiny molecules is also useful to derive the mechanism of action for unknown protein targets. (ionfieldsystems, 2011, #)
Proteases
Proteases play a significant role in the superfamily of enzymes. They regulate cellular protein activities. There are around 500 human proteases that have been identified and over 100 known homologs. An estimated data shows that up to 1200 human genes can encode proteases. In the biopharmaceutical industry, proteases are a particularly important class of protein target in terms of drug discovery screening programs. The drug discovery industry has identified highly selective and useful small molecule protease inhibitors. In disease treatment, the value of inhibitors as a drug target increases. Several environments can routinely perform screening campaigns against several protease targets. The environments being medium-throughput screening (MTS) and the most high-throughput screening (HTS).
Plasma Cleaning Of The Pipette Tips
The TipCharger system is ideal for automated screening environments. It provides automation of pipette tips cleaning that is equal to tip replacement and drastically decreases the number of tip boxes used in an assay. The reduced number of tip boxes also reduces the costs and logistical bottlenecks such as storage, delivery, and disposal.
The effectiveness of the plasma-based pipette tip is demonstrated in this study, using a fully optimized and well-characterized automated protease assay. The tip cleaning is optimized by performing plasma exposure time for each component on an individual basis for an associated peptide substrate and protease. The concluding result is compared to traditional and historical means regarding assay throughput, assay signal-to-noise ratio, and screen cost.
The Integration
The TipCharger can be used to clean pipette tips that are limited to automated liquid handlers. Exterior tips can get exposure to contaminants, which are exposed to TipCharger general plasma. They are instantly ionized. There are contaminants inside the pipette tips, which can be discharged by carrying out a series of dispense and aspirate steps. This can be done when the tips are in the cleaning station. The cleaning stations have three formats of 8, 9, and 384-well plate formats. They are a device or a consumable inside the liquid handler software. For this study, the TipCharger cleaning station is used as a reagent reservoir.
Plasma Exposure
To allow the evaluation of plasma, the protease assay setup for optimization is modified. Every column represents a particular time point in the station. The reaction plate is incubated for around twenty minutes at RT and noted by the general screening conditions. A substrate is used as a cleaning target instead of protease for a comparable test protocol.
Conclusions And Results
The results show two aspirate and cleaning of single assay reagents. Initially, the substrate is added to all the wells of the 96-well reaction plate. Protease measured to 25 ng/mL is added to column 12 of the 96-well plate as a positive control. An equal volume of a buffer is added to column 1 since six seconds of dispense steps are enough for removing the substrate of protease from pipette tips. The level to which they are removed is similar to the assay negative control. The rest of the columns of wells are used to offer a time course for pipette tip that is exposed to the plasma generated by TipCharger. These tips, which were used for the initial time course, are dirtied by dispensing and aspirating protease. After that, they are inserted into the tip cleaning stations for a time period between two to forty seconds. The same tips are then used for the aspiration of assay buffer instead of protease and it was added to a single column of the wells.
Bibliography
Ionfieldsystems. (2011). Plasma-based Cleaning of Pipette Tips in an Automated Protease Assay. Application Note AN-04, 1(2011), 1-4. http://www.ionfieldsystems.com/wp-content/uploads/2012/09/an04_protease.pdf
