AARON CIECHANOVER PRACTICAL COURSE

    First training course of the INPROTEOLYS network.

     
   Basic techniques to analyse the activity of the proteasome and ubiquitin conjugated        substrates. 1st-5th of September 2008, CICbioGUNE, Bilbao. Basque Country, Spain.

This year our course has taken the name of Aaron Ciechanover in recognition to his contributions in out field, for his outstanding help to consolidate this network and in general for supporting development of basic research.
                                     

                            Content:
                         I) Isolating proteasomes from different sources
                        II) Measuring proteasome activity in vivo and in vitro.
                       III) Purification of ubiquitinating nezymes.

                            Verifying the activity of conjugating enzymes (thiol-ester assays)

                      IV) Using in vitro ubiquitin and SUMO conjugating systems.
                            Setting up in vitro assays

With the kind participation of:

Olivier Coux

Centre de Recherche en Biochimie Macromoleculaire-CNRS, Montpellier.

Andrea Pichler

Max F. Perutz Laboratories. Medical University of Vienna Department of Medical Biochemistry. Viena

Manuel S. Rodríguez

CICbioGUNE, Bilbao. Spain.

                                          PROGRAM

DAY -1 (SATURDAY, 30th August)

           Bacterial Transformation

           GST-Hdm2, GST-SUMO-2 and GST-SUMO-3

DAY O (SUNDAY, 31st August)

Preparation of bacteria Pre-cultures

DAY 1 (MONDAY, 1st of September 2008)

8:00     Bacterial Induction (Morning)

                      Protein purification (All day)

8:30    WELCOME

9:00 INTRODUCTORY SEMINAR:                            

         Interconnecting machineries regulate the Ubiquitin- 

         Proteasome System. By Manuel S. Rodriguez.

10:00   TRAINING TO WORK WITH RADIOACTIVITY

         Content:        Security at CICbioGUNE

                              Waste recycling and processing

                              Rules for radioactivity manipulation

                              General visit

12:00   Bacterial centrifugation

12:30   Lunch

13:00 Purification of recombinant proteins using Profinia System and Home made columns

13:30 Bacterial Lysis procedure

14:00 Ultracentrifugation

14:30 In vitro Transcription and Translation of protein substrates

15:30 Binding to columns and washing procedures

17:00   Elution or binding of E3s to substrates.

19:00 Ubiquitylation Assays. Substrates: p53 and others

20:00 Evaluation of the working day

           Preparation of next day

21:00 Run gels and dry or transfer for Western-blot

DAY 2 (TUESDAY, 2nd of September 2008)

8:30     Protein purification (2nd part)

           Evaluation of the purification (Electrophoresis and Experion)

           Protein concentration and dialysis

10:00   Thiol-ester assays to check enzymes activity (cold assay)

          Ubc9, E2-25, UbcH5 .

12:00   SEMINAR:

            Regulation of SUMO and Ubiquitin conjugation at the level of E2 enzymes.

            By Andrea Pichler.   

13:00   Lunch

14:00   Run gel Thiol-ester 1hrs transfer

15:00   SUMOylation assays (cold assays)

16:00   Incubation with antibodies WB thiol-ester assays

17:00   Run gels SUMOylation assays

18:30 Tranfer O/N

19:00   Evaluation of the working day

           Preparation of next day

DAY 3 (WEDNESDAY, 3th of September 2008)

8:30  SUMOylation assays using SUMO1, SUMO2 and SUMO3 (hot assays)

         Substrates: SUMOylation of IkBa, p53 and other substrates.

9:30     Western blot for SUMO and Ubiquitin assays

           (Incubation with antibodies)

10:30 Run, dry and expose radioactive gels

12:30 Lunch

14:00 In vitro degradation assays using reticulocytes as proteasomes source

16:00 Run gels for Western-blot and/or phosphorimager detection

17:00 Introduction to the AKTA system Purifying proteasome form cell cultures (Part 1): preparation of cell extract,

ANX Sepharose and MonoQ columns

19:00   Evaluation of the working day

           Preparation of next day

DAY 4 (THURSDAY 4th of September 2008)

8:30     Purifying proteasome from cell cultures (Part 1): preparation of cell estract,

          ANX Sepharose and activity assays (screen for activity using fluorimetic assay).

12:00   SEMINAR: Structure and functions of the Proteasome.

          By Olivier Coux.

 13:00           Lunch

14:00   Purifying proteasome form cell cultures (Part 2)

           MonoQ and Hydroxyapatite columns (*).

          (*) Depending on the time, the purification on the Hydroxyapatite column might be skipped.

17:00   Activity assays, concentration of the active fractions.

19:00   Evaluation of the working day

           Preparation of next day

           Loading of the Superose 6 column.

DAY 5 (FRIDAY 5th of September 2008)

8:30     Analysis of the superose column  (activity assay)

10:00 Testing Proteasome Activity using in-gel peptidase overlay assay. (Part 1)

13:00   Lunch

14:00   Testing Proteasome Activity using in-gel peptidase overlay assys  (Part 2)

17:00 Evaluation of the working day

      Evaluation of the training course

18:00   CONCLUSIONS

           By Andrea Pichler, Olivier Coux and Manuel S. Rodríguez

20:30   Closing dinner