1. Working Group 1 Planing 1st Semester 2013

2. Planing Overview General Plan Tasks Description Deliverables: 1st Semester 2013 WG1: Recruiting Groups

3. Planing Overview Non-characterized Proteins Recombinant Proteins

4. Planing Overview Non-characterized Proteins Recombinant Proteins Universidad de Salamanca-CSIC Clones Collection ( 9800)

5. Clones Collection Clones Collection ( 9800)Clones Collection ( 13500) 13500 clones ( 2013-2014 )

6. Recombinant Proteins In situ Protein expression Conventional Protein expression Fast ●cDNA availability ●Clones sequence validated ●Yield ? Slow ●Subcloning ●Experience: E.coli, CHO, Yeast… ●High Yield ( purification ?)

7. In situ Protein Expression IVTT: Rabbits vs HeLA

8. In situ Protein Expression IVTT: Rabbits vs HeLA Optimized protocols for Protein Enrichement MS assays ( CICBiogune, UCM, ???) 25 proteins expressed by HeLa IVTT ( CIC) 50 additional clones validated ( UCM & CIC) Training of HeLa IVTT at CIC ( open)

9. Conventional Protein Expression Cloning & Subcloning strategies Selection of expression system: E.coli, CHO, yeast, Pichia,…) Non-succesfully IVTT expressed proteins High demand of recombinant proteins by other WGs Preliminary Criteria

10. Working Flow between WG1 & WG-MRM Deliverable: Data, purified protein, IVTTmix+protein, gel band, protocols,…. Amount of protein: ~ 20 µg / Lab Number of Labs for MRM: 3/protein ??? Updated list of unknown/non-characterized proteins Number of labs for MRM: Recruiting

11. Cloning No in CIC collection (9800) ( 642 chr16) No in ASU collection (13500) ( > 642 chr16?) Cloning & subcloning: After first year A few selected proteins Conventional cloning $$$ Other sources $$$$

12. Budget Scouting 600 €/ Protein HeLa IVTT for 200 µg 250€/full process per protein ( < 20 µg) Milestone: 300 proteins / 2 years 60 k€ ( personal cost no included)

13. Now is time for Suggestions Recommendations Questions Considerations More groups in WG1 etc…..

15. Work flow between WP 1 and WP-MRM -Deliverable Options: Data Protein Gel band IVTT-system Protocols -Protein validated by three labs

16. 25 proteins for MRM assays- USAL+UCM+Biogune→MRM WG 25 novel proteins in both IVTT expression systems -USAL 25 for DNA sequencing to UCM_ follow-up IVTT Heavy proteins???? Planning-options-strategy Quantity: amount 20 ug /average per protein HeLa IVTT as first option 300 proteínas en 2 años--milestone

17. Scouting 500 Euros/protein for 200 ug 200 Euros/protein full process (20 ug estimated) 300 proteins total cost= 60000 Euros/2 years

18. Antibodies

19. http://dnasu.asu.edu/DNASU/GetVectorDetail.do?vectorid=157