Thermo Fisher Scientific LiveBLAzer FRET-B/G Loading Kit with CCF2-AM

Thermo Fisher Scientific LiveBLAzer™ FRET-B/G Loading Kit with CCF2-AM

Background

GeneBLAzer® cell-based assays utilize the membrane-permeant ester forms (CCF2-AM and CCF4-AM) of the negatively charged fluorescent beta-lactamase substrates, CCF2 and CCF4. These lipophilic esters readily enter the cell, where cleavage by endogenous cytoplasmic esterases rapidly converts them into their negatively charged forms, thereby trapping them in the cytosol.

Detection of GeneBLAzer® assays is FRET-based. Each substrate is labeled with two fluorophores that form an efficient FRET pair.

• Without beta-lactamase activity: Exciting the coumarin at 409 nm in the intact CCF2 molecule results in FRET to fluorescein, emitting green fluorescence at 518 nm.
• With beta-lactamase activity: Cleavage of CCF2 separates the dyes and disrupts FRET, producing blue fluorescence at 447 nm. This blue signal can be observed under a microscope or detected as an increase in the blue channel on fluorescent microplate readers.

The CCF2-AM and CCF4-AM substrates are essential assay components for the GeneBLAzer® platform and are compatible with flow cytometry, enabling rapid clone selection. Ratiometric analysis of blue and green signals reduces well-to-well variation due to differences in cell number and substrate loading, resulting in high Z´-factor values and low coefficients of variation (CVs).

CCF2-AM and CCF4-AM differ by two carbons in the bridge linking the coumarin moiety to the lactam ring. Both are membrane-permeable, esterified forms suitable for intact cell assays.

• CCF4-AM: Better solubility (>24 hours) and improved FRET efficiency with lower background, ideal for screening applications.
• CCF2-AM: Used for general cell-based assays.

CCF2-FA is the de-esterified version of CCF2-AM, used in cell lysate applications to bypass membrane loading and de-esterification. It is preferred for assays involving cells with a cell wall and can serve as a control for acquiring excitation/emission spectra of CCF2-AM and CCF4-AM.

Specifications