96 well black flat bottom microplate

The binding of fluorescence nucleotide analog MANT (N-methylanthraniloyl)-ATP to the ILK proteins was investigated by measuring a fluorescence energy transfer signal from tryptophan of the protein to MANT-ATP. The bacterially expressed and purified ILK KLD (either WT or L207W mutant) in complex with α-Parvin CH2 was prepared at a final concentration of 1 μM in the solution (at a final volume of 80 μL) consisting of 20 mM HEPES, pH 7.5, 150 mM NaCl, 5 mM either MgCl₂ or no cation, and 4.8% glycerol in a 96-well black flat-bottom microplate (Greiner Bio-One). MANT-ATP (Life Technologies) was titrated in the reaction solution. The reaction mixtures were incubated at room temperature at 150 rpm for 10 min, and the fluorescence intensity was measured on a 2300 EnSpire Multimode Plate Reader (PerkinElmer) with excitation (280 nm) and emission (430 nm) wavelengths. The fluorescence signal was normalized by subtracting the background fluorescence of the MANT-ATP buffer, and plotted as a function of the concentration of MANT-ATP. The binding constant of MANT-ATP to ILK KLD was estimated by one-site total binding fit model using the program GraphPad Prism. The binding results were obtained in two-independent experiments performed in duplicate.

Overnight cultures of individual strains were spun down (5,000 × g, 5 min), resuspended to an OD₆₀₀ of 1.0 in PBS-T, and transferred in 500 µl aliquots into 2 ml microcentrifuge tubes. 50 µg of GFP-tagged RBP was added to the cells and incubated for 90 min on an overhead rotator. Cells were spun down and washed once with 1 ml PBS-T and resuspended in 200 µl PBS-T. 150 µl of the cell suspension was pipetted into a well of a black, flat bottom 96-well microplate (Greiner Bio-One, Austria) and the fluorescence intensity of bound GFP-RBP was measured at ambient temperature using a POLARStar Omega spectrophotometer (BMG Labtech, Germany) at 485 nm excitation, 520 nm emission with (1000 x) fixed gain. Fluorescence binding was performed in triplicate with mean (raw fluorescence) ± standard deviation. For fluorescence microscopy, 4 μl of the cell suspension was imaged using a confocal inverted microscope (Leica TCS SPE) equipped with an ACS APO 63×/1.30 oil CS lens objective with excitation at 488 nm and emissions collected with a PMT detector in the detection range of 510 to 550 nm. Transmitted-light microscopy images were obtained with the differential interference contrast mode. Images were acquired with a Leica DFC 365 FX digital camera controlled with the LAS AF software. Fiji v2.0.0 (ImageJ software) was used to generate final images.