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. Author manuscript; available in PMC: 2015 Jun 16.

Published in final edited form as: Chembiochem. 2014 May 18;15(9):1263–1267. doi: 10.1002/cbic.201402135

A) Coomassie stained 18% SDS-PAGE gel of DUB activity towards QK^UbE and QK^Ub(aux)E. Lane 1 = UCH-L3 + QK^UbE; lane 2 = UCH-L3(C95A) + QK^UbE; lane 3 = UCH-L3 + QK^Ub(aux)E; lane 4 = UCH-L3(C95A) + QK^Ub(aux)E; lane 5 = UCH-L3 + QK^Ub(aux)E + 50 mM DTT, 95 °C, 5 min. An asterisk indicates the Ub(1-76)-UCH-L3 acyl enzyme intermediate. B) ESI-MS of the observed UCH-L3-QK^Ub(aux)E adduct. Calculated m/z [M+H]⁺ 35,247.5 Da, observed 35,252.5 ± 5.6 Da.

A) Coomassie stained 18% SDS-PAGE gel of DUB activity towards QK^UbE and QK^Ub(aux)E. Lane 1 = UCH-L3 + QK^UbE; lane 2 = UCH-L3(C95A) + QK^UbE; lane 3 = UCH-L3 + QK^Ub(aux)E; lane 4 = UCH-L3(C95A) + QK^Ub(aux)E; lane 5 = UCH-L3 + QK^Ub(aux)E + 50 mM DTT, 95 °C, 5 min. An asterisk indicates the Ub(1-76)-UCH-L3 acyl enzyme intermediate. B) ESI-MS of the observed UCH-L3-QK^Ub(aux)E adduct. Calculated m/z [M+H]⁺ 35,247.5 Da, observed 35,252.5 ± 5.6 Da.