Invitrogen™ LiveBLAzer™ FRET-B/G Loading Kit with CCF2-AM

Description

• 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 endogeneous 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
• In the absence of beta-lactamase activity, exciting the coumarin at 409 nm in the intact CCF2 molecule results in FRET to the fluorescein, which emits a green fluorescence signal at 518 nm (Figure 1)
• In the presence of beta-lactamase activity, however, cleavage of CCF2 spatially separates the two dyes and disrupts FRET, so that exciting the coumarin at 409 nm now produces a blue fluorescence signal at 447 nm
• This blue signal can be readily observed under a microscope and can also be detected as an increase in the blue channel readout on fluorescent microplate readers
• The CCF2-AM and CCF4-AM substrates are essential assay components for the GeneBLAzer™ platform
• These substrates are fully compatible with flow cytometry, speeding the time to clone selection
• Ratiometric analysis of the blue and green signals reduces well-to-well variation due to differences in cell numbers and substrate loading, leading to 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 in the membrane-permeable, esterified forms, and can be used for assays in intact cells
• CCF4-AM has better solubility properties (soluble for >24 hours) than CCF2-AM and is thus best suited for screening applications
• In addition, CCF4-AM has slightly better FRET and thus slightly lower background than CCF2-AM
• CCF2-FA is essentially the CCF2 substrate without the esters found in the AM version
• CCF2-FA is de-esterified and used in cell lysate applications, bypassing loading across the cell membrane and de-esterification steps
• Cell lysates are the preferred method for applications using cells that contain a cell wall
• CCF2-FA can also be used as a control to acquire the excitation and emission spectra for CCF2-AM and CCF4-AM.