Upload
strom
View
48
Download
0
Embed Size (px)
DESCRIPTION
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. - PowerPoint PPT Presentation
Citation preview
Working Group 1Planing 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
Recombinant Proteins
Universidad de Salamanca-CSIC
Clones Collection ( 9800)
Clones Collection
Clones Collection ( 9800) Clones Collection ( 13500)
13500 clones ( 2013-2014)
Recombinant ProteinsIn situ Protein expression Conventional Protein expression
•Fast ●cDNA availability●Clones sequence validated●Yield ?
•Slow ●Subcloning●Experience: E.coli, CHO, Yeast…●High Yield ( purification ?)
In situ Protein Expression
• IVTT: Rabbits vs HeLA
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)
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
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
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 $$$ Other sources $$$$
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)
Now is time for • Suggestions • Recommendations • Questions• Considerations • More groups in WG1• etc…..
Work flow between WP 1 and WP-MRM -Deliverable Options: Data Protein Gel band IVTT-system Protocols -Protein validated by three labs
• 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
Scouting
• 500 Euros/protein for 200 ug• 200 Euros/protein full process (20 ug
estimated)• 300 proteins total cost= 60000 Euros/2 years
•
Antibodies
http://dnasu.asu.edu/DNASU/GetVectorDetail.do?vectorid=157