Small Molecule Screening

Stage I: establishment of the readout assay

• Testing reliability of the system using controls.
• Pilot screening to estimate the hit rate and criteria of hit selection (“less than 1% rule”), adjustment of library’s delivery concentration

Stage II: primary screening

• Large-scale screening of chemical library.
• Confirmation of hits, dose dependence.
• Ordering selected hits.
• Ruling out false positives (toxicity, quenching, surrogate reporter-specific, etc.)
• Running specificity assays.

Stage IV: optimizing hits, SAR

• Creating and screening the library of analogues around selected hits.
• Classification of hits, attempt to establish structure-activity relationships (SAR).
• Another phase of focused library creation and screening.

General requirements:

• Suitable for multi-well format.
• Minimal number of manipulations.
• Affordable.
• Highly reproducible with high signal-to-noise ratio.
• Availability of positive and negative controls

Success of a screening is determined at most by a robust and reliable read-out system, characterized by high assay range (link), low background, good Z-score (link). At the same time, such requirements as minimal number of manipulations and cost-effectiveness are of great importance. The Core provides assistance in the development of HTS-compatible read-out systems.

Before screen, at least 2 plates with positive and negative controls should be processed and Z-scores calculated. If Z-score<0.5, adjustments should be made to protocols/reagents/plates etc. to achieve the proper level of reproducibility.

Examples of readout systems (coming soon)

During the pilot stage of screening, set-up and optimization of screening conditions and protocols are performed. We determine the optimal concentration of drugs to be delivered (variables: the amount of drug (0.1ul or 0.2ul and well volume (min 50ul, max 250ml)) in order to provide for the desirable hit percentage (0.5-2%). In some cases, protocols should be revised to establish the appropriate step for drug addition.

• All libraries are in 96 wells plate format
• Plate design: 80 compounds per plate; 1st and 12th columns – for controls
• Molecular weight: 250 – 550
• Stock concentration of compounds: 5mg/ml (~10-20 mM) in DMSO
• Compound delivery: hanging drop (0.1uL or 0.2uL)
• Reaction volume: 50-250uL per well
• Final concentration of compounds: ~ 4 - 40uM

The Core provides assistance in data processing to select statistically significant "hits" ( see Protocols/Tutorials section), as well as in "hit" characterization and search for similar compounds available in the Core and on the market. The Core can help to establish contacts with outside partners to create focused sub-libraries of small molecules around a "hit" and to design a program for the chemical optimization of lead compounds.

SMSC is opening a bank of common-use antibodies. Currently we have a set of secondary antibodies shown below; each is conjugated with DyLight 488 and DyLight 594 fluorescent dyes (Jackson ImmunoResearch Laboratories). The antibodies work well in place of fluorescein/Alexa488 & Texas Red conjugates (see the picture below).

To purchase antibodies, please fill in the Antibody Request Form and e-mail it to shRNA@ccf.org

DyLight 488, DyLight 594	aliquot	price
Goat Anti-Mouse IgG(H+L)    ML* (min X Hu, Bov, Hrs, Rb, Sw Sr Prot)	50ul	$12
Goat Anti-Mouse IgG, Fcy Subclass 1 specific      ML* (min X Hu, Bov, Rb Sr Prot)	25ul	$24
Goat Anti-Rabbit IgG(H+L)      ML* (min X Hu, Ms, Rat Sr Prot)	50ul	$12
Goat Anti-Rat IgG(H+L)      ML* (min X Hu, Bov, Hrs,Rb Sr Prot)	50ul	$12
Donkey Anti-Goat IgG(H+L)      ML* (min X Ck,GP,Sy Hms,Hrs,Hu,Ms,Rb,Rat Sr Prot)	25ul	$18
Donkey Anti-Guineea Pig IgG(H+L)      ML* (min X Bov, Ck,Gt,Sy Hms, Hrs,Hu,Ms, Rb,Rat,Shp Sr Prot)	25ul	$18
Normal Goat Serum	1 ml	$6
Normal Donkey Serum	1 ml	$6
A set of all aliquots (2 colors of each secondary abs+both serums) $180

The Small Molecule Screening Core (SMSC) has negotiated an opportunity to order shRNA clones from Sigma (in the form of glycerol stocks) for LRI researchers for a very low price on a per clone basis. To order, please fill in the New shRNA order form.
To order clones from the SMSC shRNA collections (see below), please fill in the
Old shRNA order form and e-mail it to shRNA@ccf.org
If you do not have CCF activity #, please fill out the
Credit Card Authorization Form

- Orders from the SMSC shRNA collection are available in 1-3 days;
- Orders for new shRNA are places on the 20th of each month, and will be available in ~ 3 weeks after the placement.

The SMSC has a growing collection of almost 3000 shRNA clones (Sigma-Aldrich), in the form of glycerol stocks. New clones are added every two months.

http://www.sigmaaldrich.com/life-science/functional-genomics-and-rnai/shrna.html

The list of available clones with controls (bacterial glycerol stocks):

• 2012 LRI shRNA CLONE LIST

The clones are sequenced-verified shRNA lentiviral plasmids (pLKO.1-puro) provided as frozen bacterial glycerol stocks (Terrific Broth, carbenicillin at 100 µg/ml and 10% glycerol).

http://www.sigmaaldrich.com/life-science/functional-genomics-and-rnai/shrna/library-information/vector-map.html

The MISSION pLKO.1-puro Control Vector is a lentivirus plasmid vector. The vector does not contain an shRNA insert and is useful as a negative control in experiments using the MISSION shRNA library clones. This allows one to examine the effect of transfection on gene expression and interpret the knockdown effect seen with shRNA clones. Ampicillin and puromycin antibiotic resistance genes provide selection in bacterial or mammalian cells respectively. In addition, self-inactivating replication incompetent viral particles can be produced in packaging cells (HEK293T) by co-transfection with compatible packaging plasmids.

The MISSION eGFP shRNA Control Vector is a 7,091 base pair lentivirus plasmid vector that contains an shRNA sequence targeting eGFP (GenBank Accession No. pEGFP U55761).1 The eGFP shRNA Control Vector is useful as a positive knockdown control in experiments using cell lines expressing eGFP.

The MISSION Luciferase shRNA Control Vector is a 7,091 base pair lentivirus plasmid vector that contains an shRNA sequence targeting luciferase from Photinus pyralis (GenBank Accession No. M15077). The Luciferase shRNA Control Vector is useful as a positive knockdown control in experiments using cell lines expressing firefly luciferase.