Journal: Journal of proteome research

Article Title: Automated High-Throughput Affinity Capture-Mass Spectrometry Platform with Data-Independent Acquisition.

doi: 10.1021/acs.jproteome.4c00696

Figure Lengend Snippet: Figure 1. Magnetic bead-based automatable AC-MS chemoproteomics workflow. (A) Structures of Cp19-PEG2-Affi-Gel 10 and Cp19-PEG4/8/ 12-biotin. The nonselective kinase inhibitor Cp19 moiety is highlighted in purple, and the Affi-Gel 10 or biotin affinity moiety is highlighted in green. (B) Western blot (WB) detection of BTK, TBK1, and CDK2 enrichment by Cp19-PEG4/8/12-biotin using SA-Mag beads in comparison to that by Cp19-PEG2-Affi-Gel 10 from K562 cell lysates. The two black triangles point to isoforms 1 and 2 of CDK2. (C) Schematic overview of the automated AC-MS chemoproteomics workflow. The time that each step takes for a 96-well plate of samples is indicated. Highlighted in red is the time for the steps that mainly require manual preparation. O/N stands for overnight.

Article Snippet: The membrane was blocked with Intercept (TBS) blocking buffer (927-60001, Licor) at room temperature for 1 h and incubated with primary antibody BTK mAb (1:1000, 8547, Cell Signaling), TBK1 mAb (1:1000, 3504, Cell Signaling), or CDK2 (1:1000, 2546, Cell Signaling) in Intercept T20 (TBS) antibody diluent (927-65001, Licor) overnight at 4 °C, followed by the IRDye 800CW Goat anti-Rabbit IgG secondary antibody (1:5000, 926-32211, Licor) at room temperature for 1 h. After several washes with TBST, images were acquired by an ODYSSEY DLx imaging system (Licor).

Techniques: Western Blot, Comparison