Quantification of Ubiquitinated PCNA

^TAMRA−ubiquitinated PCNA was prepared as described34 (link) and mixed to USP4 CD and FL at the described concentrations. Aliquots of the reaction were loaded on SDS–polyacrylamide gel electrophoresis after being stopped with loading buffer at different time points. Bands corresponding to ^TAMRA−ubiquitinated PCNA (S) and ^TAMRA−ubiquitin (P) were quantified using ChemiDoc XSR (Bio-Rad). Product concentration was calculated as S₀ · (P/(P+S)), where S₀ is the initial substrate concentration.

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Clerici M., Luna-Vargas M.P., Faesen A.C, & Sixma T.K. (2014). The DUSP–Ubl domain of USP4 enhances its catalytic efficiency by promoting ubiquitin exchange. Nature Communications, 5, 5399.

Publication 2014

ChemiDoc XSR

Buffer Pcna Sds polyacrylamide gel electrophoresis Ubiquitin

Corresponding Organization :

Other organizations : The Netherlands Cancer Institute

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Variable analysis

independent variables

Concentration of USP4 CD (catalytic domain)
Concentration of USP4 FL (full length)

dependent variables

Concentration of TAMRA-ubiquitinated PCNA (S)
Concentration of TAMRA-ubiquitin (P)

control variables

TAMRA-ubiquitinated PCNA prepared as described in reference 34
Reaction stopped with loading buffer at different time points

controls

Positive control: TAMRA-ubiquitinated PCNA prepared as described in reference 34
Negative control: Not explicitly mentioned

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