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Working Group 1 Planing 1st Semester 2013

Working Group 1 Planing 1st Semester 2013. Planing Overview. General Plan Tasks Description Deliverables : 1st Semester 2013 WG1: Recruiting Groups. Planing Overview. Non- characterized Proteins. Recombinant Proteins. Planing Overview. Non- characterized Proteins.

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Working Group 1 Planing 1st Semester 2013

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  1. WorkingGroup1 Planing 1st Semester 2013

  2. PlaningOverview • General Plan • TasksDescription • Deliverables: 1st Semester 2013 • WG1: RecruitingGroups

  3. PlaningOverview Non-characterizedProteins RecombinantProteins

  4. PlaningOverview Non-characterizedProteins RecombinantProteins Universidad de Salamanca-CSIC Clones Collection ( 9800)

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

  6. RecombinantProteins In situ Proteinexpression ConventionalProteinexpression •Fast ●cDNAavailability ●Clones sequencevalidated ●Yield ? •Slow ●Subcloning ●Experience: E.coli, CHO, Yeast… ●HighYield ( purification ?)

  7. In situ ProteinExpression • IVTT: Rabbits vs HeLA

  8. In situ ProteinExpression • IVTT: Rabbits vs HeLA • OptimizedprotocolsforProteinEnrichement • MS assays ( CICBiogune, UCM, ???) • 25 proteinsexpressedbyHeLa IVTT ( CIC) • 50 additional clones validated ( UCM & CIC) • Training of HeLa IVTT at CIC ( open)

  9. ConventionalProteinExpression PreliminaryCriteria • Cloning & Subcloningstrategies • Selection of expressionsystem: E.coli, CHO, yeast, Pichia,…) • Non-succesfully IVTT expressedproteins • Highdemand of recombinantproteinsbyotherWGs

  10. WorkingFlowbetweenWG1 & WG-MRM • Deliverable: Data, purifiedprotein, IVTTmix+protein, gel band, protocols,…. • Amount of protein: ~ 20 µg / Lab • Number of Labsfor MRM: 3/protein ??? • Updatedlist of unknown/non-characterizedproteins • Number of labsfor MRM: Recruiting

  11. Cloning • No in CIC collection (9800) ( 642 chr16) • No in ASU collection (13500) ( > 642 chr16?) • Cloning & subcloning: Afterfirstyear A fewselectedproteins Conventionalcloning $$$ Othersources $$$$

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

  13. Nowis time for • Suggestions • Recommendations • Questions • Considerations • More groups in WG1 • etc…..

  14. Workflowbetween WP 1 and WP-MRM -DeliverableOptions: Data Protein Gel band IVTT-system Protocols -Proteinvalidatedbythreelabs

  15. 25 proteinsfor MRM assays-USAL+UCM+Biogune→MRM WG • 25 novel proteins in both IVTT expressionsystems -USAL • 25 for DNA sequencingto UCM_ follow-up IVTT • Heavy proteins???? Planning-options-strategy • Quantity: amount 20 ug /average per protein HeLa IVTT as firstoption • 300 proteínas en 2 años--milestone

  16. Scouting • 500 Euros/proteinfor 200 ug • 200 Euros/protein full process (20 ugestimated) • 300 proteins total cost= 60000 Euros/2 years

  17. Antibodies

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

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