Warscheid Lab - In vitro kinase assay of hFLNc domain 18-21

In vitro kinase assay of human FLNc domain 18-21 with Akt and PKC alpha

  • Organism: Homo sapiens
  • Instrument: LTQ Orbitrap Elite
  • SpikeIn: No
  • Keywords: FLNc, in vitro kinase assay, AKT, PKCa
Abstract
In vitro kinase assay with human filamin C domain 18-21 recombinantly expressed in E.coli, purified by Ni2+-NTA beads and incubated with PKC alpha, Akt or a combination of both to identify kinase specific phosphorylation sites.
Experiment Description
Recombinantly expressed and purified mouse and human FLNc d18-21 WT was dialyzed overnight at 4°C in dialysis buffer (1 mM DTT, 100 mM KCl, 20 mM HEPES pH 7.4, 10 mM MgCl2). For MS-coupled kinase assays, H2O was added to 100 µg protein to a total volume of 200 µl and mixed with 10 x kinase buffer (NEB, Frankfurt, Germany). The assay was started by adding 200 ng Akt (Proteinkinase, Kassel, Germany) and/or 200 ng PKC alpha (Sigma-Aldrich) in the presence of 1x PKC lipid activator (Merck Millipore, Darmstadt, Germany). The reaction was carried out for 20 min at 30°C and 200 rpm. One tenth of each of the three independent replicates was used for gel-based analyses. The remaining sample was diluted 1:4 (v/v) with 50 mM ammonium bicarbonate and subjected to in-solution digestion using sequencing grade trypsin (1:50) (Promega) for 3.5 h at 42°C and 200 rpm on a thermoshaker. Single protein digests were acidified with TFA [final concentration 1% (v/v)], subjected to TiO2-based phosphopeptide enrichment, and analyzed by LC-MS/MS using MSA, HCD and ETD fragmentation.
Created on 4/10/19, 10:46 AM
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Figure 4A. 

In vitro kinase assay coupled to phosphorylation-dependent mobility shift analysis. Reactions were performed using recombinant hFLNc d18-21 WT in the presence (+) or absence (-) of ATP, Akt and/or PKCα as indicated. Immunoblot analysis performed with an antibody directed against the EEF-tag that was fused to the carboxy-terminus of hFLNc d18-21 WT revealed multiple shifted bands, indicating kinase-dependent phosphorylation events. h, human; WT, wild-type

Figure 4B. 

In vitro kinase assay coupled to quantitative MS analysis for site determination. Reactions were performed as described in figure 4A. MS data for each kinase were quantified using Skyline and are available in this Panorama project. Intensities of phosphopeptides distinctive for a specific phosphorylation site in hFLNc d18-21 WT were added up per experiment and represented as normalized mean ± SEM (n=3).