| Assay Method Information | |
| | Biochemical Assays |
| Description: | iFLiK and HTRF studies were carried out as described in Z. Fang, J. R. Simard, D. Plenker, H. D. Nguyen, T. Phan, P. Wolle, S. Baumeister, D. Rauh, ACS Chem. Biol. 2015, 10, 279-288.All reagents for HTRF experiments were purchased from Cisbio Bioassays, France. OriginPro 9.1G software (OriginLab Corporation, Northhampton, Mass.) was used for data analysis and data was fit to a sigmoidal dose-response model using the following four-parameter logistic equation:y = A 2 + ( A 2 - A 1 ) ( 1 + ( x IC 50 ) p ) ( 1 ) (A1: bottom asymptote; A2: top asymptote; IC50: half-maximal inhibitory concentration; p: Hill coefficient) Kinetic Characterization of Covalent Probe Compounds:Time-dependent IC50 measurements were performed with activated full-length Akt1 as described under Biochemical assays. Briefly, IC50 values were determined for twelve different incubation times and afterwards plotted versus accordingly. Data was analyzed according to literature procedure as described in B. F. Krippendorff, R. Neuhaus, P. Lienau, A. Reichel, W. Huisinga, J. Biomol. Screen. 2009, 14, 913-923. Ki and kinact were calculated with XLfit (Version 5.4.0.8, IDBS, Munich, Germany) defining the substrate concentration as 250 nM and the corresponding substrate KM as 150 nM.Mass Spectrometry:Purified full-length wtAkt1 was thawed under cold water and diluted to a final concentration of 1 mg/mL in storage buffer (50 mM HEPES, 200 mM NaCl, 10% Glycerol, pH 7.4). 20 μL of the respective mixture were mixed with 2 molar equivalents of the compounds of formulas (1a) and (2a), respectively (10 mM in DMSO); samples containing equal volumes of DMSO were individually prepared for control measurements. Following incubation for thirty minutes on ice, the samples were analyzed by ESI-MS using an Agilent 1100 Series HPLC System connected to a ThermoFinnigan LTQ Linear Ion Trap mass spectrometer. Therefore, 6 μL of sample were injected and separated using a Vydac 214TP C4 5 u column (150 mm×2.1 mm) starting at 20% of solvent B for five minutes followed by a gradient up to 90% of solvent B over 14 min (flow rate 210 μL/min) with 0.1% TFA in water as solvent A and 0.1% TFA in acetonitrile as solvent B. After washing the column for two minutes with 90% of solvent B, the concentration of solvent A was increased to 80% in 1 min and the column was washed for five additional minutes. During the complete experiment, a mass range of 700 to 2000 m/z was scanned and raw data was deconvoluted and analyzed with MagTran and mMass (Version 5.5.0) software.For ESI-MS/MS measurements, samples were denatured, separated via SDS-PAGE followed by staining with Coomassie Brilliant Blue and prepared according to standard tryptic in-gel digest protocols as described in A. Shevchenko, H. Tomas, J. Havlis, J. V. Olsen, M. Mann, Nat. Protoc. 2006, 1, 2856-2860. Subsequently, samples were thawed, dissolved in 20 μL of 0.1% TFA in water, sonicated at room temperature for 15 min, and centrifuged at 15000×g for 1 min shortly before analysis. 3 μL of sample were loaded onto a pre-column cartridge and desalted for 5 min using 0.1% TFA in water as eluent at a flow rate of 30 μL/min. The samples were back-flushed from the pre-column to the nano-HPLC column during the whole analysis. Elution was performed using a gradient starting at 5% B with a final composition of 30% B after 35 min (flow rate 300 nL/min) using 0.1% formic acid in water as eluent A and 0.1% formic acid in acetonitrile as eluent B and a column temperature of 40° C. The nano-HPLC column was washed by increasing the percentage of solvent B to 60% in 5 min and to 95% in additional 5 min, washing the columns for further 5 min, flushing back to starting conditions and equilibration of the system for 14 min. During the complete gradient cycle, a typical TOP10 shot-gun proteomics method for the MS and MS/MS analysis was used. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |