Labeling and Purification of Proteins

UBQLN2 proteins were expressed, purified and, as indicated, labeled with Alexa Fluor 647, Alexa Fluor 555, or DyLight-488 as described previously (15 (link)). Substrates were purified by Ni-NTA chromatography and SUMO protease cleavage as described previously, and R-Neh2Dual-ACTR-DHFR was labeled with sulfo-cyanine5 maleimide (Lumiprobe) on a single unique cysteine immediately N-terminal to DHFR, and repurified by gel filtration as described previously (26 (link), 34 (link)). 26S Proteasome was purified from S. cerevisiae via a 3X-FLAG tag on Rpn11 as described previously (26 (link)). AMSH*, oTUB1* and vOTU were purified as described previously (32 (link)). vOTU was labeled with Alexa Fluor 555 and repurified by gel filtration. Ub₄ was synthesized and purified as described previously (22 ). Substrate, UBQLN2 and DUB protein sequences are given in Supplemental Table S2

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Valentino I.M., Llivicota-Guaman J.G., Dao T.P., Mulvey E.O., Lehman A.M., Galagedera S.K., Mallon E.L., Castañeda C.A, & Kraut D.A. (2024). Phase separation of polyubiquitinated proteins in UBQLN2 condensates controls substrate fate. bioRxiv.

Publication Preprint 2024

sulfo-cyanine5 maleimide

Corresponding Organization : Syracuse University

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Variable analysis

independent variables

Labeling of UBQLN2 proteins with Alexa Fluor 647, Alexa Fluor 555, or DyLight-488
Labeling of R-Neh2Dual-ACTR-DHFR with sulfo-cyanine5 maleimide
Purification of UBQLN2 proteins, substrates, 26S Proteasome, AMSH*, oTUB1*, and vOTU

dependent variables

Not explicitly mentioned

control variables

Purification of 26S Proteasome via a 3X-FLAG tag on Rpn11
Purification of AMSH*, oTUB1*, and vOTU as described previously

positive controls

None mentioned

negative controls

None mentioned

Annotations

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