Katina Lazarow / Martin Neuenschwander

Project application for biochemical high-throughput screening

First step:

Contact us via E-Mail: --> see Contact

(if you do not receive any answer within 24 hours please also give us a call)

After initial discussions, we will ask you to fill out our questionnaire.

The questionnaire will help us in planning the project and serves as a reference document. It is also a kind of checklist.

Before starting the primary screening, we will require a signed legal agreement besides an official order. 

Assay Setup
Depending on the complexity of the assay, you either may send us the materials (like target protein) or visit us at the Screening Unit, where we transfer the assay to the 384-well format and test the assay set up for HTS usage. You will then be able to update the project application form with the revised protocol. With a working assay protocol at hand, the material & reagent costs for primary screening plus validation are calculated and a quote is generated.

Pilot and Primary Screen
After receiving the order, we perform the primary screen either with one person from your team or on our own. The generated data is processed using in-house generated software and KNIME workflows. After completion of the primary screening step, you receive a data package containing the raw data, quality control plots, statistic analysis, and proposed hit lists containing also structural data. Primary screening is conducted usually at a compound concentration of 10 uM. The pilot screen usually encompasses 9 library plates with FDA-approved drugs, run in 2 technical replicates to asses assay reproducibility. The primary screen is run in 1 technical replicate.

Review of hit lists
If desired, you may revise the hit lists.

Optional: Reconfirmation and counter screen at 10 uM

If the number of primary actives is in the range of 352 to 1056, up to 1056 compounds can be repicked into assay ready plates in two technical replicates using our acoustic dispenser. Optionally, a counter screening condition can be added. This reconfirmation screening step is effective in removing random false positives (and some of the chemical false positives if a counter screen is applied).

Cherry picking

We generate a 384-well plate containing your requested hits for validation. 5 ul of a 10 mM solution are picked at our compound management facility, usually the maximum number here is 352 compounds.

IC₅₀ validation and optional counter screen
IC₅₀ validation (using nine to ten serial dilutions of the compounds in two technical replicates) is carried out by us, the data are analyzed using R scripts and KNIME workflows, curve fits are generated automatically using an algorithm with automated outlier detection and robust curve fitting. At this step also a concentration-dependent counter screen can be applied.

Quality control of up to 352 hit compounds via LC-MS Analysis
A small aliquot (0.2 µl) of your hit picking plate is taken and diluted in ACN/H₂O for the LC-MS analysis at our compound management facility. We analyze the samples in ACN/H₂O (1:1, 25 µM) with our Agilent TOF mass spectrometer and determine the purity via UV absorption at 254 nm.

Review of IC₅₀ data

You receive IC₅₀ reports containing MS analysis data, frequent hitter information, and reference data from biophysical and cytotox profiling. Our chemists revise with you the list of validated compounds and suggest what compounds might be selected for further characterization and optimization of leads.

Residual compounds on the picked plate can be provided to your laboratory for validation assays.

Optionally we offer also Surface Plasmon Resonance validation of the hits if applicable.

The Surface Plasmon Resonance detection system allows automated screening for compounds (even 100 Dalton sized) which bind to proteins immobilized on chips. Different types of chip surfaces allow to couple proteins under mild conditions via His- or GSH- tags, by antibodies or strepravidine.
Specific biological activity of proteins must be controlled by defined binders and non-binders to monitor stability of proteins after repeated regeneration steps for removal of nonspecifically binding compounds.
Kd, plus On and Off rates can be calculated from kinetic data.

The service can be used to characterize small molecules that emerged as hit compounds from a screening project, but also user-provided compounds can be tested.