Module Director: Jack Saari
206 543-5633
jsaari@u.washington.edu
A. Description
The Biochemistry/Immunology module provides NEI-funded investigators at the University of Washington with the following services: (1) instrumentation for biochemical and morphological procedures, (2) preparation and characterization of monoclonal antibodies (mAbs), (3) provision of specialized antibodies generated in house, (4) localization of mRNAs in tissue sections by in situ hybridization, (5) single and double-label immunocytochemistry in both sections and whole mounts, (6) characterization of antibodies by Western, ELISA, and immunocytochemical analyses, determination of IgG subclass, (7) isolation of IgG fractions from ascites, or hybridoma cell culture supernatants. These services enhance the capability of NEI-funded investigators to carry out vision research by providing expertise and equipment not currently available in their laboratories and by providing custom monoclonal antibody production at a fraction of the cost of commercial services. In addition, the shared used of resources (equipment and technical expertise) extends the impact of increasingly scarce resource funds. Jing Huang, MD, B/I Module Research Scientist since 1987, will continue to perform module services during the next project period. She has demonstrated proficiency in all services offered.
Services:
Monoclonal Antibody Production. The module technician will immunize the mice, verify a polyclonal antibody response, fuse mouse myeloma cells with cells from the spleen of the immunized mouse, screen hybridoma cells via ELISA, Western analysis, or immunocytochemistry, clone hybridoma cells from soft agar or by limiting dilution, type the antibody, produce ascites, and isolate an IgG fraction. The investigator may be asked to assist during the screening, depending on the method used. The investigator will supply the antigen for immunization and screening and the mice. The procedure takes 10-12 weeks from immunization of the mice to isolation of a mAb from ascites.
Localization of mRNAs via in situ hybridization. The module technician will prepare DIG (digoxigenin)-labeled probes from vectors, prepare tissue sections or blocks from suitably fixed tissue, incubate probes with tissues, process for observation of the results, and prepare images for publications or slides. The investigator will supply cDNA or other vectors suitable for production of probes.
Single and/or double label immunocytochemistry. The module technician will fix the tissue, cut cryosections, probe with one or two antibodies, and/or DAPI, and prepare images for publication and/or slides. Immunocytochemistry of whole mounts is also offered.
Apoptosis assays. Cells undergoing apoptosis are detected with the TUNEL (terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling) assay. The current methodology makes use of the ApopTag® (Serologicals Corporation). Antibodies to Caspases 3 or 8 can also be used to detect apoptotic cells.
Antigen retrieval with paraffin sections. Sections are deparaffinized, rehydrated, and heated in a microwave oven for various times/temperatures to unmask antigens. The conditions must be optimized for each antigen and antibody.
Characterization of antibodies. The module technician will determine the type of immunoglobulin (e.g., IgM or G, and the subtype), and will examine the specificity of the antibody via Western analysis, ELISA, immunocytochemistry or immunoprecipitation.
Weaning hybridoma cell lines. The module technician will wean hybridoma cell lines from serum-containing media to serum-free media. This procedure facilitates the purification of IgG fractions from cell culture supernatants and is useful for high-density cell culture production methods using hollow-fiber bioreactors.
Location:
Module equipment is housed in several rooms. RR-808 is a wet laboratory that houses an incubator, 4-foot biological safety cabinet, and microscopes and is used for production of monoclonal antibodies, preparation of sections for immunocytochemistry, cell culture, and for in situ hybridization. RR-820 is a wet laboratory that houses equipment items shared by several investigators including centrifuges, liquid scintillation counters, incubators, and –80o C freezers. RR-819 is a walk-in cold room with sink and running water, which is used for protein/enzyme preparations. It is equipped with red illumination and a light-tight door for preparation of light sensitive materials such as rhodopsin and retinoid-binding proteins. A Nikon fluorescence microscope is housed in RR-834, a room shared with the Imaging Module. A 7-foot biological safety cabinet is housed in RR-841, a room devoted to cell culture. A detailed description of the services and equipment available is provided below.
B. History of the B/I Module
The Biochemistry Module and Cell Culture/Immunology Modules were created as independent units in 1976 when CORE funding was initiated at the University of Washington. In 1989, the two modules were combined under the direction of Drs Saari (Director) and Beavo (Co-Director). Dr. Saari will continue as Module director and Dr Hurley will become Co-Director for the next project period. Jing Huang¸MD, joined the CORE in 1987 and will continue to be responsible for the day-to-day activities of the module. She was promoted to Research Scientist in 2003. Over the years, the mission of the module has undergone continual evolution in response to the changing needs of NEI-supported investigators. In recent years, there has been substantial demand for production of monoclonal antibodies (mAbs), for single- and double-label immunocytochemistry, and for in situ hybridization, and we will continue to offer these services during the next project period.
In response to a growing demand from NEI-funded investigators, we propose to add protein identification by mass spectrometry (proteomics) to the services available from the B/I Module.
