Upstream Workshop on Ubiquitin and SUMO
Dates: December 3-7, 2012
Organizers: Koraljka Husnjak, Ivan Dikic
Location: Institute of Biochemistry II, Goethe University,
Frankfurt am Main, Germany
Frankfurt am Main, Germany
December 2012
Tutors/teachers
Masato Akutsu
Anna Gottfried
Koraljka Husnjak
Katarzyna Kliza
Stefan Müller
Simin Rahighi
Rebecca Ullmann
Sjoerd van Wijk
Participants
Nitin Jindal, ES
Beata Jonik-Nowak, FR
Mona Moghimi, NL
Aysegul Sapmaz, NL
Jón Otti Sigurdsson, DK
Katharina Witting, NL
Katarzyna Kliza, DE
Experiments
1. In vitro ubiquitination (Katarzyna Kliza, Koraljka Husnjak)
2. In vitro deubiquitination (Katarzyna Kliza, Koraljka Husnjak)
3. Synthesis and purification of K48-, K63- and K-11linked ubiquitin chains (Masato
Akutsu)
4. In vivo ubiquitination assays (Katarzyna Kliza, Koraljka Husnjak)
5. Non-covalent binding of ubiquitin and ubiquitin-like proteins to their binding
domains (Simin Rahighi, Koraljka Husnjak, Katarzyna Kliza)
6. In vitro sumoylation (Rebecca Ullmann, Anna Gottfried)
7. In vivo sumoylation (Rebecca Ullmann, Anna Gottfried)
Lectures
Ubiquitin and ubiquitin networks
Koraljka Husnjak
Small ubiquitin-related modifier
Stefan Müller
Structural aspects of linkage recognition by various ubiquitin-binding domains
How to quantify UBD:Ub(l) interactions?
What determines the specificity of linkage formation and disassembly?
Simin Rahighi
Deubiquitinating enzymes – structure and function
Masato Akutsu
Fluorescence-based sensors to monitor localization and functions of various
ubiquitin chains in cells
Sjoerd van Wijk
Participants divided in three groups
Group 1 (Nitin Jindal, Beata Jonik-Nowak)
Group 2 (Mona Moghimi, Jón Otti Sigurdsson)
Group 3 (Aysegul Sapmaz, Katharina Witting)
Course agenda (daily 9-18 h, with lunch break 13-14 h)
Sunday, December 2, 2012
 Arrival of participants
 Tour around the Institute of Biochemistry II
 Welcome dinner
Monday, December 3, 2012
 Short introductory talk (Koraljka Husnjak)
 Safety talk for Institute of Biochemistry II (Stefanie Oess)
 Transient transfection of cells (Exp 4, 5)
 Preparation of SDS-PAGE gels
 SDS-PAGE and Coomassie staining (to evaluate quality of recombinant
proteins used in experiments)
 Serum-starvation of cells (Exp 4 - EGFR)
 Discussion
Tuesday, December 4, 2012
 EGF stimulation (Exp 4, EGFR)
 Lysis of cells (Exp 4 - EGFR, NEMO, Exp 5)
 Setup of immunoprecipitation (Exp 4 - EGFR)
 Setup of GST pulldown assays (Exp 5)
 Washing of immunoprecipitation/GST pulldown samples
 Preparation of samples for SDS-PAGE (Exp 4, 5)
 Preparation of SDS-PAGE gels (Exp 4, 5)
 Transient transfection of cells (Exp 7)
 Discussion
Wednesday, December 5, 2012
 SDS-PAGE and gel transfer (Exp 4, 5)
 In vitro ubiquitination assays (Exp 1)
 Synthesis of K48-, K63- and K-11 linked ubiquitin chains (Exp 3)
 Small ubiquitin-related modifier (Stefan Müller)
 In vitro sumoylation reaction (Exp 6)
 Lysis of cells and setup of immunoprecipitation (Exp 7)
 Discussion
Thursday, December 6, 2012
 Immunoblotting (Exp 4, 5)
 Ubiquitin chain purification (MonoS column, AKTA) (Exp 3)
 In vitro deubiquitination (Exp 2)
 Preparation of SDS-PAGE gels (Exp 2, 3, 6, 7)
 SDS-PAGE and gel transfer (Exp 2, 3, 6, 7)
 Ubiquitin and ubiquitin networks (Koraljka Husnjak)
 Discussion
Friday, December 7, 2012
 Immunoblotting (Exp 2, 3, 6, 7)
 Structural aspects of linkage recognition by various ubiquitin-binding
domains. How to quantify UBD:Ub(l) interactions? What determines the
specificity of linkage formation and disassembly? (Simin Rahighi)
 Deubiquitinating enzymes – structure and function (Masato Akutsu)
 Fluorescence-based sensors to monitor localization and functions of various
ubiquitin chains in cells (Sjoerd van Wijk)
 General discussion
 Concluding remarks
 Farewell lunch