The Zebrafish Protein & Antibody Core will take the first formal step in creating a national resource for the production of zebrafish proteins and their respective antibodies for use by the zebrafish research community. Effort will center on three efforts: 1) production of zebrafish proteins and protein fragments; 2) production of antibody fragments; and 3) the fabrication of arrayed proteins and affinity reagents. Zebrafish proteins targeted for expression would include transcription factors, enzymes, cytoskeletal proteins, cell signaling proteins, growth factors, ectodomains of receptors, etc. Wherever possible, full-length versions of the proteins would be expressed, but due to the difficulty of working with certain protein classes (e.g., membrane proteins, multi-domain proteins), their expression would be limited to soluble domains. Expression would be achieved primarily in E. coli, with insect cell systems used for high priority targets. For the first year, a variety of different protein classes would be targeted so that we may develop protocols relevant to a wide range of proteins; we would aim for the expression of 100 proteins, and the generation of antibodies to 50 targets. During the second year, we would ramp up the production scale of targets and antibodies by 50%, and phase in proteins nominated by the community at large. It is anticipated that the number of user requests would double during the third year, for a total of 200 proteins and 100 antibodies. In addition, we would fabricate arrays of proteins and antibodies for distribution during the third year. To oversee this pilot project, there will be a scientific advisory committee composed of zebrafish scientists, and a technical advisory committee composed of biotechnologists. We propose a sharing plan consisting of a website containing publicly accessible data, presentations at scientific meetings, and distribution of requested reagents and protocols. ? ? ?
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| Pershad, Kritika; Sullivan, Mark A; Kay, Brian K (2011) Drop-out phagemid vector for switching from phage displayed affinity reagents to expression formats. Anal Biochem 412:210-6 |
| Kay, Brian K; Thai, Sang; Volgina, Veronica V (2009) High-throughput biotinylation of proteins. Methods Mol Biol 498:185-96 |
