Inhibition of ARC4/tankyrase binding motif interaction
EOS300131
Description
The assay is based on the interaction of the human TNKS2 protein binding domain ARC4 with a tankyrase binding motif consisting of 8 amino acids. The ARC4 domain is produced as fusion protein with cyan fluorescent protein (CFP, variant mCerulean), while the tankyrase binding motif is produced as fusion protein with yellow fluorescent protein (YFP, variant mCitrine). Proteins are recombinantly produced in E. coli in Oulu.
Upon interaction, the CFP is brought into proximity of the YFP, resulting in a Förster-resonance-energy transfer (FRET) from excitation of CFP at 430 nm resulting in emission from YFP at 527 nm. We therefore measure the fluorescence intensity of the emission of CFP (477 nm, donor emission) and YFP (527 nm, acceptor emission) upon CFP excitation at 430 nm. We calculate the ratiometric FRET signal (YFP emission:CFP emission, r430) after fluorescence readout and blank subtraction for each well.
Assay setup
Assay stage
Bioassay
Bioassay type
Bioassay setting
Assay organism
BAO tags
Detection method
Physical detection method
Detection instrument
Synergy Neo2 Multi-Mode Reader (BioTek)
Target
Attachments
Something else
Activities
Concentration unit
Activity determination method
Ratio of FRET to donor emission is calculated for two excitation wavelengths, 430 nm and 410 nm.
For some run batches, the ratio values are adjusted to the medians by plate row prior to further normalization. This was done in order to account for observed spatial effects caused by dispensing.
The 430 nm ratio (r430) is normalized to the means of the positive and negative control groups, setting the positive control to 100% and the negative control to 0%. Positive controls contain a high salt solution that inhibits protein association and reduces FRET, while negative control solutions exhibit FRET due to protein association.
A compound is considered active if it has a normalized r430 (r430%) greater than 20% and does not exhibit fluorescence interference. A compound is considered inconclusive if the r430% is greater than 20% and it exhibits fluorescence interference. Fluorescence interference was determined using two filters: 1) the donor emission from excitation at 430 nm had to fall within a range determined by the controls on each plate (50%+/- the mean donor emission of controls) and 2) the percentage difference between the r410 and r430 values could not exceed 50%. Percentage difference was calculated as 100%*|r410-r430|/(0.5*(r410+r430)).
Compounds exhibiting r430% less than or equal to 20% were considered inactive. Compounds that show fluorescence interference and r430% > 20% are considered inconclusive.