SPR Binding Assay for MDM2-UbcH5B-Ub Interaction

SPR binding experiments were performed at 25 °C on a Biacore T200 instrument using a CM-5 chip (GE Healthcare) with coupled anti-GST antibody as described previously.²⁰ (link) Briefly, GST-tagged MDM2 variants were coupled on the chip and a serial dilution of UbcH5B–Ub in running buffer containing 25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1 mM DTT and 0.005% (v/v) Tween-20 was used as analyte. Two technical replicates were performed and data were analyzed with BIAevalution (GE Healthcare) and Scrubber2 (BioLogic Software).

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Magnussen H.M, & Huang D.T. (2021). Identification of a Catalytic Active but Non-Aggregating MDM2 RING Domain Variant. Journal of Molecular Biology, 433(5), 166807.

Publication 2021

Biacore T200 CM-5 chip BIAevalution

Anti antibody Biologic Buffer Chip Dilution Mdm2 Nacl T200 Tris Tween 20

Corresponding Organization : Cancer Research UK

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Variable analysis

independent variables

Concentration of UbcH5B–Ub

dependent variables

SPR binding

control variables

Temperature (25 °C)
Buffer composition (25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1 mM DTT, 0.005% (v/v) Tween-20)
Instrument (Biacore T200)
Chip (CM-5 chip with coupled anti-GST antibody)
GST-tagged MDM2 variants coupled on the chip

controls

Two technical replicates

Annotations

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