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| Kinasera-FRET live cell assay |
ARC-Fluo probes can be easily conjugated with bright and photostable fuorescent labels that are optically suitable for cellular assays and offer the maximal suitability with the fluorescent protein used as FRET donor.
The assay can be performed in the 96-well microplate format with 40-100 microL detection volumes. Attached cells expressing the kinase under investigation as a fusion with a fluorescent protein (e.g. AcGFP1 or DsRed-Monomer) are treated with ARC-Fluo probe (labeled with fluorescent dye e.g. TAMRA or Alexa647) for 60 minutes to enable the uptake of the probe. The cells are washed to remove the excess of the probe and nutrient mixture. The change in the FRET signal between the donor (fluorescent protein) and acceptor dye (ARC-Fluo) upon stimulation or inhibition of the cells can be monitored in real time using conventional plate reader or fluorescent microscope.
A Schematic illustration of PKA activation: The activation of adenylate cyclases (AC) by Forskolin induces the synthesis of cAMP. Activation of PKA by cAMP leads to the dissociation of PKA holoenzyme and the released free active PKAc-YFP binds ARC-Photo probe that brings together the YFP donor fuorophore and the acceptor fuorophore of the ARC-Photo probe, resulting in increased energy transfer between the fuorophores. This effect is reversed by competitive inhibitors (H89) that competitively displaces ARC-Fluo probe from the complex.
The assay can be performed using either a fluorescent microscope or a platereader. Either the decrease in the donor emission or increase in acceptor emission can be monitored or FRET can be calculated when dual wavelength detection is performed.
Monitoring the activity of PKA in live cells. CHO cells over-expressing PKA-YFP were grown to 80% confluence on black 96-well cell culture plates, incubated with ARC-Fluo probe for 60 minutes in serum free Ham's F-12 nutrient mixture, washed twice with HBSS. (A) The emission intensity of the donor ?uorophore (em. 540 nm) in cells in the presence (solid line) or absence (dotted line) of ARC-1042 (acceptor) n = 10 and (B) the emission intensity of the acceptor ARC-1042 (em. 605 nm) in cells expressing (solid line) or not expressing (dotted line) PKAc-YFP (donor) n = 7 upon excitation at 500 nm determined with a fluorescence microscope. (C) Detection of FRET changes in living cells using a fluorescence microplate reader. FRET efficiency between the donor fluorophore of PKAc-YFP and the acceptor dye of the ARC-Photo probe was measured as the ratio of fluorescence emission intensities (590/520) in C9H6 cells non-treated (dotted line) or treated (solid line) with ARC-Fluo porbe.