Selection and Production of Recombinant Binders for Use in Protein Microarrays

Nonfiction, Health & Well Being, Medical, Medical Science, Microbiology
Cover of the book Selection and Production of Recombinant Binders for Use in Protein Microarrays by Mark Laible, GRIN Verlag
View on Amazon View on AbeBooks View on Kobo View on B.Depository View on eBay View on Walmart
Author: Mark Laible ISBN: 9783640994267
Publisher: GRIN Verlag Publication: August 29, 2011
Imprint: GRIN Verlag Language: English
Author: Mark Laible
ISBN: 9783640994267
Publisher: GRIN Verlag
Publication: August 29, 2011
Imprint: GRIN Verlag
Language: English

Diploma Thesis from the year 2009 in the subject Biology - Micro- and Molecular Biology, grade: 1,1, German Center of Cancer Research Heidelberg, language: English, abstract: Antibodys are essential everyday tools for molecular biologists and much depends on their performance and availabilty. Large numbers of antibodys are available already, however, these cover only a small part of the known proteom. The generation of antibodies for proteom-wide studies using conventional methods, which are based on animal immunisation, would be hardly feasible and not sustainable. Recombinant binders may be able to fill the gap of missing affinity reagents. The main advantages of recombinant binders are (1) the wide availabilty in form of fully synthetic DNA libraries (2) the independance on animal immunisation, which still is the main source of antibodies today and (3) the possibility to design completely new classes of synthetic binders with definded properties. The aim of this work was to establish the selection and purification of recombinant binders for use in protein microarrays. Binders were selected from a synthetic library of single chain fragment variable (scFv) antibodies using phage display as tool for selection. A number of binders against gamma globulins and BSA-FITC could be selected from the library and were subsequently used for establishment of downstream applications. Several methods for expression and purification of the binders were tested mostly in regard to scFv yield. Methods for detection were evaluated for their sensitivity and specificity. As the long term aim was the setup of a protein microarray based on recombinant binders, multiple immobilisation strategies were evaluated, including a one step on-chip purification and immobilisation of scFvs from crude expression samples. The on-chip purification was shown to work. However, the best results regarding signal intensity were obtained using biotinylated protein-A purified scFvs immobilised in a directed manner on streptavidin-coated glass slides. Undirected immobilisation of purified scFvs on epoxy-silane slides was as well shown to be a possibility. In a side project, the novel phage RDA (representational difference analysis) technique for PCR and DNA hybridisation based selection of binders was applied. The major focus was put onto optimizing the critical steps of amplification of the scFv library by PCR.

View on Amazon View on AbeBooks View on Kobo View on B.Depository View on eBay View on Walmart

Diploma Thesis from the year 2009 in the subject Biology - Micro- and Molecular Biology, grade: 1,1, German Center of Cancer Research Heidelberg, language: English, abstract: Antibodys are essential everyday tools for molecular biologists and much depends on their performance and availabilty. Large numbers of antibodys are available already, however, these cover only a small part of the known proteom. The generation of antibodies for proteom-wide studies using conventional methods, which are based on animal immunisation, would be hardly feasible and not sustainable. Recombinant binders may be able to fill the gap of missing affinity reagents. The main advantages of recombinant binders are (1) the wide availabilty in form of fully synthetic DNA libraries (2) the independance on animal immunisation, which still is the main source of antibodies today and (3) the possibility to design completely new classes of synthetic binders with definded properties. The aim of this work was to establish the selection and purification of recombinant binders for use in protein microarrays. Binders were selected from a synthetic library of single chain fragment variable (scFv) antibodies using phage display as tool for selection. A number of binders against gamma globulins and BSA-FITC could be selected from the library and were subsequently used for establishment of downstream applications. Several methods for expression and purification of the binders were tested mostly in regard to scFv yield. Methods for detection were evaluated for their sensitivity and specificity. As the long term aim was the setup of a protein microarray based on recombinant binders, multiple immobilisation strategies were evaluated, including a one step on-chip purification and immobilisation of scFvs from crude expression samples. The on-chip purification was shown to work. However, the best results regarding signal intensity were obtained using biotinylated protein-A purified scFvs immobilised in a directed manner on streptavidin-coated glass slides. Undirected immobilisation of purified scFvs on epoxy-silane slides was as well shown to be a possibility. In a side project, the novel phage RDA (representational difference analysis) technique for PCR and DNA hybridisation based selection of binders was applied. The major focus was put onto optimizing the critical steps of amplification of the scFv library by PCR.

More books from GRIN Verlag

Cover of the book Von der GHH zur Neuen Mitte - Nutzungswandel auf einer zentralen Fläche in Oberhausen by Mark Laible
Cover of the book Sprachentwicklung und Sprachverständnis und ihre Störungen by Mark Laible
Cover of the book Wirkung von Daily Soaps auf Jugendliche und die Anschlusskommunikation im Internet by Mark Laible
Cover of the book Der eschatologische Aspekt in der Bedeutung des Sukkot-Festes by Mark Laible
Cover of the book Die Schopenhauerrezeption und ihre Funktion in Eugenio Cambaceres` 'Sin rumbo' by Mark Laible
Cover of the book Unternehmensmitbestimmung by Mark Laible
Cover of the book Habitualisiertes und impulsives Kaufverhalten by Mark Laible
Cover of the book Schwebende Rechtsstreitigkeiten in der Rechnungslegung nach IAS/IFRS by Mark Laible
Cover of the book Berechnung der Vergütung, insbesondere Vermögenswirksame Leistungen (Unterweisung Verwaltungsfachangestellte / -r) by Mark Laible
Cover of the book Siemens in China - Joint Ventures by Mark Laible
Cover of the book Die Identität des Ostseeraums by Mark Laible
Cover of the book Ernst Rudolf Huber im Nationalsozialismus und im Geschichtsunterricht by Mark Laible
Cover of the book Metaphor and metonymy in the conception of emotion in different cultures by Mark Laible
Cover of the book Diskriminanzanalysen im Rahmen von Bilanzanalysen zur Bonitätsbewertung by Mark Laible
Cover of the book Holismus und Artenschutz by Mark Laible
We use our own "cookies" and third party cookies to improve services and to see statistical information. By using this website, you agree to our Privacy Policy