Fluorescent Labeling of Human AurA Kinase

Protein samples were prepared using a cysteine-light construct of human AurA expressed in BL21-DE3-RIL cells (Agilent) and purified as described (28 (link), 65 (link)). Cysteine residues were incorporated at L225 on the D-helix and S284 on the activation loop for labeling with Alexa 488 maleimide (Thermo Fisher) as the donor, and Alexa 568 maleimide as the acceptor. Labeling of donor-only (D-O) and donor-plus-acceptor samples (D+A) was verified by mass spectrometry (SI Appendix, Fig. S1 B and C). A synthetic construct of human Tpx2 (residues 1–43; Selleckchem or Genscript) or a recombinant construct of GST-tagged Tpx2 (residues 1–43) was used for experiments requiring Tpx2.

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Lake E.W., Muretta J.M., Thompson A.R., Rasmussen D.M., Majumdar A., Faber E.B., Ruff E.F., Thomas D.D, & Levinson N.M. (2018). Quantitative conformational profiling of kinase inhibitors reveals origins of selectivity for Aurora kinase activation states. Proceedings of the National Academy of Sciences of the United States of America, 115(51), E11894-E11903.

Publication 2018

BL21-DE3-RIL cells Alexa 488 maleimide

Alexa 568 Cells Cysteine Donor Helix Human Light Maleimide Mass spectrometry Protein Tpx2

Corresponding Organization :

Other organizations : University of Minnesota, University of Minnesota Medical Center

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Variable analysis

independent variables

Cysteine-light construct of human AurA expressed in BL21-DE3-RIL cells
Labeling with Alexa 488 maleimide (donor) and Alexa 568 maleimide (acceptor)
Use of synthetic construct of human Tpx2 (residues 1-43) or recombinant construct of GST-tagged Tpx2 (residues 1-43)

dependent variables

Measured outcomes not explicitly mentioned

control variables

Cysteine residues incorporated at L225 on the D-helix and S284 on the activation loop
Labeling of donor-only (D-O) and donor-plus-acceptor samples (D+A) verified by mass spectrometry

positive controls

Donor-only (D-O) samples

negative controls

Donor-plus-acceptor (D+A) samples

Annotations

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