Condition Scouting: Probe 96 Experimental Conditions in a Single Run

Each channel of the CFM can be a different solution, enabling up to 96 different experimental conditions to be probed in a single experiment.

Because the ligand deposition process is done using the CFM’s enclosed microchannels, each ligand spot can have a different concentration, buffer composition, pH, detergent mix, and so forth. Thus, 96 different conditions can be assayed in a single step, while the protein of interest is still active.

For biosensor work this could include the optimization of:

  • Buffer composition
  • Coupling conditions
  • Capture conditions
  • Solubilization detergents
  • Regeneration conditions

For example, in prior work studying GPCR solubilization conditions, we optimized the solubilization of CXCR5 in 2 hours by evaluating 96 different solubilization cocktails, while the same process would have up to a week on a standard SPR biosensor and 96 changes of the system running buffer.

CFM_8

FURTHER READING

PUBLICATION: Detergent screening of a G-protein-coupled receptor using serial and array biosensor technologies

Anal Biochem. 2009 Mar 1;386(1):98-104. doi: 10.1016/j.ab.2008.12.011. Epub 2008 Dec 24.

Rich RL1, Miles AR, Gale BK, Myszka DG.
1Center for Biomolecular Interaction Analysis, School of Medicine, University of Utah, Salt Lake City, 84132, USA.

We describe the benefits and limitations of two biosensor approaches for screening solubilization conditions for G-protein-coupled receptors (GPCRs). Assays designed for a serial processing instrument (Biacore 2000/3000/T100) and an array platform (Biacore Flexchip) were used to examine how effectively 96 different detergents solubilized the chemokine receptor CCR5 while maintaining its binding activity for a conformationally sensitive Fab (2D7). Using the serial processing instrument, we were able to analyze three samples in each 30-min binding cycle, thereby requiring approximately 24h to screen an entire 96-well plate of conditions. In-line capturing allowed us to normalize the 2D7 binding responses for different receptor capture levels. In contrast, with the array system, we could characterize the effects of all 96 detergents simultaneously, completing the assay in less than 1h. But the current array technology requires that we capture the GPCR preparations off-line, making it more challenging to normalize for receptor capture levels. Also, the array platform is less sensitive than the serial platforms, thereby limiting the size of the analyte to larger molecules (>5000Da). Overall, the two approaches proved to be highly complementary; both assays identified identical detergents that produced active solubilized CCR5 as well as those detergents that either were ineffective solubilizers or inactivated the receptor.

GO TO PUB>

[vc_widget_sidebar sidebar_id=”applications-submenu”]