The UW Small Molecule Screening Facility collaborates with academic investigators to perform high-throughput screens of chemical libraries. Our compound collection includes over 100,000 small molecules, including commercially available compound libraries and unique natural product libraries. Data generated from screens are entered into a screening results database. Access to this non-public database is granted only to SMSF investigators and collaborators who have deposited data. The screening facility personnel assist efforts by providing access to the compound libraries, and by operating and maintaining the screening robots. Collaborators perform their own assays and provide their own reagents. To allow us to prioritize new screening projects appropriately, a detailed application must be submitted in order to initiate a screen at the UW-SMSF. Additional applications must be submitted each time a new screen is proposed.
Radioactive assays are currently not supported in the screening facility, and substances requiring a higher containment level than BSL2 are not permitted.
Assay Volume and Plate Type
For a high-throughput screen, a biochemical or cell-based assay must be adapted to a microtiter plate format. Assays are most commonly performed in 384-well assay plates; in certain cases, they may also be performed in 96-well or 1536-well. Assay volumes in 384-well plates range from 5 µl (in low-volume plates) to 100 µl (in standard plates). At the low end of this range, inaccuracies in small-volume pipetting can cause signal variation, while there is a risk of spillage and cross-contamination at the high end.
Different types of plates are recommended, depending on the assay detection method used. White plates are recommended for luminescence assays, black plates for fluorescence assays, clear-bottom plates for automated microscopy, and clear polystyrene plates for photometric assays.
Replicates and Controls
Most high-throughput assays show a high amount of inherent variability and error. Control readings are essential to a well-designed assay, and every assay should use as many controls as possible. In general, there are two types of controls: plate-based controls and assay-wide controls. Plate-based controls are controls that are placed on each individual assay plate. These are essential for identifying plate-to-plate variability and establishing assay background levels. Assays that are prone to plate-wise variability (such as luciferase readouts that decay over time) should primarily use plate-based controls and normalization. Stock library plates are formatted with empty wells to allow for screen-specific plate-based controls.
Signal/background ratio (S/B), and well-to-well variability (CV) are important issues to consider. As assay variability increases, the S/B ratio must increase for the screen to be successful. We recommend using a positive control condition to determine the S/B ratio. To determine S/B, fill a plate with reagents using the same equipment to be used for the screen. Add several dilutions of the positive control to several wells, and determine whether the positive control can be reproducibly detected above the well-to-well variation. These data will provide an indication of the false-positive and false-negative rate of your assay.
Quantitative Assay Evaluation and Optimization
The Z´-factor calculation is useful during piloting for quality assessment of assay conditions (Zhang et. al. 1999, see below for full reference). An assay can be considered validated for high-throughput screening after 3 independent experiments (for example, each experiment set up separately starting from scratch or carried out on different days) have been shown to result in reproducible and suitable Z´-factor values.
To quantitatively rank assay conditions, perform control experiments and calculate Z´ from the data collected:
SD + = positive control standard deviation
SD - = negative control standard deviation
Ave + = positive control average
Ave - = negative control average
Note – Microsoft Excel has a STDEV function that works well for this calculation
After calculating Z´ the following table is useful for evaluating the potential performance you might expect using this assay to screen at the UW screening facility. If optimization is needed, different assay conditions should be compared and ranked by their Z´-factor values until suitable conditions are found.
High-throughput Screening Assay Fitness Table
1 > Z´ > 0.75 An excellent assay
0.75 > Z´ > 0.5 A good assay
0.5 = Z´ The absolute minimum recommend for high throughput screening
Z'-factor Reference: Ji-Hu Zhang, Thomas D. Y. Chung and Kevin R. Oldenburg (1999). A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J. Biomol. Screen 4:67-73.
Performing a Screen
Once a satisfactory assay is developed in an assay plate format, follow the online appointment directions to schedule your screen. Screening facility personnel will provide screeners with training in the use of walk-up instruments to be used during the course of your screen. Only screening facility personnel are permitted to handle library stock plates, and some screening equipment and thus they perform all transfers from library plates into assay plates. After establishing an assay protocol, you should perform a pilot screen of 1200 wells. If the results of your pilot screen are satisfactory, you may continue to screen additional library plates. When performing a screen, it is preferable to process as many plates as possible during a single visit to the screening facility. Libraries contain 13-57 plates in total. All consumables are supplied by the SMSF and will be charged to your account after use. Reagents can also be ordered and shipped directly to SMSF, and will also be added to your bill.
Raw screening data should be saved directly to your UW SMSF server folder. SMSF staff will load your data into the SMSF database. Once data are entered into the database, instruction will be provided as to how to view your data and compare your results to those of other screens.
Selection of compounds for secondary assays ("cherry picking") is permitted only after deposition of initial data.
Small Molecule Compounds for Secondary Screens
After completing a primary screen of a library, you should have many screening "positives" to follow-up in secondary screens. Subject to compound availability, we will provide you with 1 µl of each selected screening positive compound (~5 µg). These are called “cherry picks”. The maximum number of cherry picks allowed is equal to 0.3% of the number of compounds initially screened. For example, if the ChemBridge library is the initial screen completed (16,320 compounds), a maximum of 50 cherry picks can be requested. With adequate justification, exceptions to this policy can be made.
Screeners with a "hit" rate that is higher than 0.3% will often choose their cherry picks by examining the structures of their screening positives and comparing these data with others' screening data. This requires data entry into the screening database and also time spent by the researcher mining the database. This step is usually important for the success of the project.
If you require more than 1 µl of compound, you must order it directly from the original source. Generally, 1-5 mg of a compound can be purchased for less than $50 from the manufacturer for commercial libraries. Commercial sources are often able to resupply a majority of their compounds for the first year after a library is purchased. The resupply rate falls for older libraries
For compounds no longer available from the original vendor, other supply sources may be found using SciFinder or emolecules.com.