Lecturer: Angela Koehler
Instructors: Noreen Lyell, Leslie McClain and Becky Meyer
TAs: Colin Kim and Shelbi Parker
Lab manager: Hsinhwa Lee
Chemical probes, or ligands, are important research tools used to explore cellular processes and therapeutic targets. The use of high-throughput and unbiased strategies to identify small molecules that bind specific biomolecules, such as proteins, can provide insight on the structure or function of targets. Additionally, a small-molecule screen can identify new chemical probes for target proteins of interest.
The small-molecule microarray (SMM) is a high-throughput method that enables the detection of protein-ligand binding. Briefly, ligands are 'printed' onto a slide and incubated with purifed protein. Unbound protein is washed from the slide and bound protein is detected using a tag on the protein of interest. Because the location of every ligand on the slide is known, the detection of protein indicates that it is bound to the ligand at that location.
In your experiment, you build upon work completed by students in the Sp17, Sp18, and Sp19 semesters! An SMM was completed in Sp17 and secondary assays were used to examine putative hits in Sp18 and Sp19. The secondary assays were in vitro experiments that focused on protein activity and ligand binding. You will contribute to this project by completing an in vivo assay that examines the impact of ligand binding on the stability of the protein of interest.
This module has been developed thanks to the generous time and thoughtful efforts of several Koehler Laboratory members, in particular Nick Struntz and Rob Wilson.
Lab links: day by day
M3D1: Prepare for cellular thermal shift assay
M3D2: Incubate with ligand and apply heat treatment for protein denaturation
M3D3: Begin Western blot analysis
M3D4: Complete antibody staining for Western blot analysis
M3D5: Data analysis and assignment preparation
Research proposal presentations
Notes for teaching faculty
Prep notes for M3