Gene Delivery to Mammalian Cells Vol 2: Viral Gene Transfer Techniques (Methods in Molecular Biology Vol 246)

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Science , — Scott, J. Smith, G. Barbas III, C.

Hoogenboom, H. Human antibodies from synthetic repertoires of germline VH-gene segments rearranged in vitro. USA 89 , — Janda, K. USA 91 , — Devlin, J. Bass, S.

Proteins 8 , — Rebar, E. Crameri, R. Allergy Immunol. Borrebaeck C. Today 19 , — High Throughput Screen 2 , 63— Kay, B. A laboratory manual. Academic Press, San Diego. Primer sequences and GenBank accession numbers for the resulting constructs are listed in Table S1. Phagemid rescue from E. Recombinant phages were amplified from E. Virion assembly was monitored by spot titration as described [25].

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The tubes were then incubated for 1 h at RT on a rotating wheel. Thereafter, the beads were washed 3x by first immobilizing the beads by using a Dynal tube magnet rack. The supernatant was discarded and 0. The tubes were taken out of the rack and briefly vortexed before re-entered into the rack. The supernatant was again cleared and the washing repeated twice. The tubes were then incubated for 1. The tubes were washed 3x in PBST as described above. In one experiment, the WB buffer pH 8 was supplemented with mM imidazole.

Virion assembly and functionality analysis of pVII modified helper phages. A Virion assembly efficiency in E. Normal denoted as wt and pVII tag-modified M13K07 helper phage production was assessed by infectious titration, and values given as the number of kanamycin-resistant kanR colony forming units cfu per ml culture supernatant. B Phagemid rescue ability of normal and modified M13K07 helper phages.


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C Phagemid ampR to helper phage kanR ratio in infectious titration. The bead capture was done as described by the manufacturer protocol in the recommended sample volumes as described in Methods. Binding and subsequent detection of captured virions were done with an anti-M13 Ab as described in Methods. Moreover, virion capture was most efficient when done at pH 8.

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GenBank accession numbers and QuikChange mutagenesis primers. The authors would like to thank Sathiyaruby Manikam Vadivelu for excellent technical assistance, Professor Norbert Roos for conducting the electron microscopy analysis and Dr. Herald Reiersen for helpful discussions. Conceived and designed the experiments: GL. Performed the experiments: GL. Analyzed the data: GL IS. Wrote the paper: GL IS. Browse Subject Areas?

Click through the PLOS taxonomy to find articles in your field. Abstract Background Phage display is a platform for selection of specific binding molecules and this is a clear-cut motivation for increasing its performance. Introduction Phage display is a platform for selection of binders with affinity for specific target molecules, and also exhibits high versatility with respect to target discovery [1].

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Download: PPT. Figure 1. Discussion Heterologous peptide display on pIII or pVIII is based on signal sequence dependent translocation of the fusion from cytosol to the periplasm. Virion production Phagemid rescue from E.

Organic Chemistry 51C. Lecture 18. Amino Acids, Peptides, and Proteins.

Supporting Information. Figure S1. Figure S2. Figure S3. Figure S4. Table S1. Acknowledgments The authors would like to thank Sathiyaruby Manikam Vadivelu for excellent technical assistance, Professor Norbert Roos for conducting the electron microscopy analysis and Dr. Author Contributions Conceived and designed the experiments: GL. References 1. Bratkovic T Progress in phage display: evolution of the technique and its applications. Cell Mol Life Sci — View Article Google Scholar 2. Methods Enzymol — View Article Google Scholar 3. Hoogenboom HR Selecting and screening recombinant antibody libraries.

Nat Biotechnol — Find it at other libraries via WorldCat Limited preview. Contributor Kay, Brian K. Winter, Jill. McCafferty, John, Dr. Bibliography Includes bibliographical references and index. Contents Biology of the filamentous bacteriophage, B. Webster-- principles and applications of phage display, B.

Kay and R. Hoess-- vectors for phage display, N. Adey, S. McConnell, and B. Kay-- construction of random peptide libraries in bacteriophage M13, A. Sparks, N. Adey, J. Beasley, and B. Kay-- construction and screening of antibody display libraries, J. McCafferty and K. Johnson-- phagemid-displayed peptide libraries, D. Dottavio and J. Winter-- phage libraries displaying random peptides derived from a target sequence, D. Jordaan-- display and selection of proteins on genetic packages, R.

Suter, M.


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