Synthesis and Characterization of Probes

All commercial chemicals and solvents were purchased from Sigma Aldrich or Fisher Scientific and used without further purification. The identity and purity of each product was characterized by MS, HPLC, TLC, and NMR. Purity of target compounds has been determined to be >95% by LC/MS on a Waters Autopurification system with PDA, MicroMass ZQ and ELSD detector and a reversed phase column (Waters X-Bridge C18, 4.6 × 150 mm, 5 μm) eluted with water/acetonitrile gradients, containing 0.1% TFA. Stock solutions of all inhibitors were prepared in molecular biology grade DMSO (Sigma Aldrich) at 1,000× concentrations. The PU-TCO, PU-CW800 and YK5-B probes and relevant control probes, and the PU-beads and the control probes were generated using published protocols^{13 (link),19 (link),35 (link),80 (link)–85 (link)} or as described in Supplementary Notes 1. The GA-biotin probe was purchased from Sigma (SML0985). Disuccinimidyl suberate (DSS) was acquired from ThermoFisher (21655).

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McNutt S.W., Roychowdhury T., Pasala C., Nguyen H.T., Thornton D.T., Sharma S., Botticelli L., Digwal C.S., Joshi S., Yang N., Panchal P., Chakrabarty S., Bay S., Markov V., Kwong C., Lisanti J., Chung S.Y., Ginsberg S.D., Yan P., DeStanchina E., Corben A., Modi S., Alpaugh M., Colombo G., Erdjument-Bromage H., Neubert T.A., Chalkley R.J., Baker P.R., Burlingame A.L., Rodina A., Chiosis G, & Chu F. (2024). Phosphorylation-Driven Epichaperome Assembly: A Critical Regulator of Cellular Adaptability and Proliferation. Research Square.

Publication Preprint 2024

Autopurification system MicroMass ZQ X-Bridge C18 molecular biology grade DMSO Disuccinimidyl suberate (DSS)

Corresponding Organization :

Other organizations : Memorial Sloan Kettering Cancer Center, University of New Hampshire, University of New Hampshire at Manchester, Harvard University, Nathan Kline Institute for Psychiatric Research, University of Pavia, Neurosciences Institute, University of California, San Francisco, University of California System

Top 5 similar protocols

Variable analysis

independent variables

Concentration of inhibitors

dependent variables

Purity of target compounds
Characterization of identity and purity of each product by MS, HPLC, TLC, and NMR

control variables

All commercial chemicals and solvents were purchased from Sigma Aldrich or Fisher Scientific and used without further purification
Stock solutions of all inhibitors were prepared in molecular biology grade DMSO (Sigma Aldrich) at 1,000× concentrations
The PU-TCO, PU-CW800 and YK5-B probes and relevant control probes, and the PU-beads and the control probes were generated using published protocols
The GA-biotin probe was purchased from Sigma (SML0985)
Disuccinimidyl suberate (DSS) was acquired from ThermoFisher (21655)

positive controls

Relevant control probes for the PU-TCO, PU-CW800 and YK5-B probes, and the PU-beads

negative controls

Control probes for the PU-TCO, PU-CW800 and YK5-B probes, and the PU-beads

Annotations

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