Instructions: Please fill information in Section 1 and return to the KU-HTS contact Melinda Broward
( mbroward@kumc.edu
). Information in Section 2 will be completed in meeting to be scheduled with
Principal Investigator and KU-HTS personnel .
SECTION 1
Principal Investigator Information
Name:
Institution:
Department/Lab:
Screen Charge to information (charge code or bill to information):
E-mail:
Name:
Phone#:
Fax #:
Personnel working on project (name and position):
Assay, Rationale, and Technology of the Screen
Provide a brief description of the biology, rationale, and goals for conducting the screen.
Describe the assay materials and methods
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If you would like to add additional information on biology, scientific rationale for the target, preliminary data etc., please attach your write up to this document. Limit attachment to NOT more than 5 pages.
SECTION 2
This Section to be completed during consultation meeting with KU-HTS personnel.
Assay Protocol
If commercial kits are available, provide brief description.
Manufacturer name and Catalog #:
General Assay parameters to be optimized:
Throughput mode
False positive/negative rates (historic information)
Sensitivity of the assay
Signal window
Cell seeding process
Coefficient of variation (CV)
Z and Z’ values
Cost of reagents during scale-up
Assay Signal Stability
Miniaturization of the Assay in microplate format:
A. Biochemical Assays
Volume adjustment: 70-90ul (384-wells) or 100-200 ul (96-well)
Assay sensitivity and specificity
Order of reagent addition
DMSO sensitivity
Substrate/Ligand concentration
Incubation time and degree of substrate depletion
Windows between positive and negative controls
Assay reproducibility (2 plates, 2 days)
IC50 curves of known compounds
Enzyme/substrate stability, storage, freeze-thaw effects
B. Cell-based Assays
Cell cycle and cell-passage dependence
Cell numbers at seeding in 384-well and 96-well formats
Time course of read-out
Cell harvesting conditions
Signal-background ratios
Incubation time with test compounds: pre-incubation possibities
Minimized pipetting steps, washing steps and no. of buffers
Influence of the order of reagent addition/possibility of pooling reagents
Try to use the same model of instruments (e.g. detection and liquid handling) for both assay development and screening, if possible.
Check reagents for screening
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Scaling-up for HTS
Identification of vendor/contractor for outsourcing reagent preparation
Negotiating Reagent costs
Large-scale protein production
Optimization of parameters for large-scale cell-growth (pH, media, confluence, CO
2 levels, detachment, osmolarity
(ion-channel targets only)
Tips on improving expression, minimize edge effects, cellaggregation
Original completed form goes to HTS file and electronic copy to Project Director Melinda Broward at mbroward@kumc.edu
.
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