Raf-1 Alpha SureFire CETSA Target Engagement Assay Kit, 500 assay points

Alpha SureFire^® CETSA^® Target Engagement kits efficiently assess target engagement in live cells, on endogenously expressed target protein with no need for engineered protein constructs or cell lines to gain confidence with earlier, better prediction in the drug discovery process.

Benefits:

• Cell-based assay for target engagement using all cell types (primary cells, cell lines, iPS etc.)
• Utilized as physiologically relevant model with endogenously expressed target protein (no need for cloning)
• Detection based on rapid, simple Alpha technology (no washing or separation steps)
• Validated protocol by Pelago Bioscience
• Optimized cell lysis buffers (selection of five buffers for assay development)
• Minimal handling in a streamlined workflow, for fastest results
• Excellent reproducibility for consistent outcomes
• Robotics- and automation-compatible for increased productivity

The Cellular Thermal Shift Assay (CETSA^®) assesses the thermal stability of proteins in living cells and cell lysate based on denaturation and aggregation upon heating. The relative amount of remaining soluble protein after heating is measured, and a thermal melting curve of target protein is generated. Compound binding affects the thermal stability of the protein, with a shift in the melting curve indicative of cellular target engagement.

CETSA^® is performed by incubating intact cells with the test compound. A melting curve is generated where the sample, in the presence and in the absence of a reference compound, is heat challenged at 12 different temperatures. From the melting curve, a single temperature is selected where there is an obvious shift of the reference compound. This single temperature is then used to perform single concentration or concentration-response curve compound screening. An alternative assay format is to run the assay with disrupted cells. This is useful in measuring the ability of the compound to engage the target protein independently of its ability to cross the cell membrane, and of its potential efflux or metabolization by the cells.

Alpha SureFire CETSA^® assay kits enable the rapid, sensitive, and quantitative detection of target protein that remains soluble after heat treatment of compound-treated cells without any need for separating soluble and aggregated protein (no washing steps; no separation steps). In an Alpha SureFire CETSA^® assay, one antibody against target protein binds to streptavidin-coated Alpha Donor beads, while a second antibody against the target is bound through a proprietary CaptSure™ agent to AlphaLISA Acceptor beads. In the presence of the target protein, the two antibodies bring the Donor and Acceptor Beads close to each other, enabling the generation of an Alpha signal upon illumination of Donor Beads by the Alpha-enabled plate reader, such as the EnVision^®, EnSight^™, or VICTOR^® Nivo^™ multimode plate readers. The amount of light emission is directly proportional to the amount of soluble protein present in the sample.

This assay eliminates the need for laborious techniques, such as Western blotting or conventional ELISA. Alpha SureFire CETSA^® is a homogeneous assay with no sample washing steps required, allowing for minimal handling, short assay times, better well-to-well reproducibility (lower CV%), and robotic operation if desired.

Formats:

• The 500-point kit contains enough reagents to run 500 wells in 384-well format using a 20 µL reaction volume, and contains small bottles of each of the five types of lysis buffer.
• The 5,000-point kits contain enough reagents to run 5,000 wells in 384-well format using a 20 µL reaction volume. Each 5,000 point kit contains a different cell lysis buffer:
  • CETSA-RAF-1-A5K1 contains CETSA^® Cell Lysis Buffer 1
  • CETSA-RAF-1-A5K2 contains CETSA^® Cell Lysis Buffer 2
  • CETSA- RAF-1-A5K3 contains CETSA^® Cell Lysis Buffer 3
  • CETSA- RAF-1-A5K4 contains CETSA^® Cell Lysis Buffer 4
  • CETSA-RAF-1-A5K5 contains CETSA^® Cell Lysis Buffer 5