The Technical Core will provide three broad functions that will assist the four Projects of the PPG. These are Protein Purification, Protein Expression Analysis, and Gene Expression Analysis. The components of these are outlined below, and include both support personnel and equipment. The Technical Core will facilitate the progress of each project as described below, as well ss interaction between the five labs comprising the Program Project. 1. Protein purification: FPLC. The Perkins currently has an FPLC for protein purification, and this will become part of the Technical Support Core. This will be utilized by Krause's project (Project 2) to purify the factor that binds to the upstream regulatory region of the CD34 promoter. It will also be used by Weissman's project (Specific Aim 4) to identify the proteins that bind to upstream regulatory sequences of genes that are down- regulated on neutrophil activation. Since the FPLC is already on site, we only requesting additional columns that are needed. 2. Protein expression analysis A. 2d-GELS: Equipment,, supplies, technician (Dr. Zheng) and Melanie II software B. MALDI-MS: Technician )Edward Papacoda). A key overriding theme to this PPG is the analysis of the Molecular Anatomy of hematopoietic cells during the process of myelopoiesis, including the initial stages (Project 2), as well as in induced maturation in normal and EVI-1-expressing cells (Project 3), and in response to E. coli (Project 4). These projects will examine changes in both the mRNA level (see next section) and the protein level. One of the most exciting new developments in molecular biology is the ability to identify proteins by tryptic digest and mass spectrometry, using the MALDI-ms (matrix-assisted laser desorption/ionization mass spectrometry) in conjunction with the Prosite software for searching protein and DNA sequence databases. This has allowed the identification of proteins using as little as 10 fmol, which is less than 1 ng of a 50,000 dalton protein (equivalent to the lower limit of what is detectable on silver-stained gels). In parallel, hardware and software for 2D gel analysis of proteins has improved, to allow reproducible fractionation of proteins, and detailed comparison of different gels to identify quantitative differences. We intend to take full advantage of these advances, and are requesting money for equipment, supplies, and technical support to enable us to do this. The cumulative accrual of information and expertise that will occur with this core facility will substantially benefit all of the projects in this PPG, and provides a cogent argument in its support. 3. Gene expression analysis A. Affymetrix gene chip technology B. Differential Display 3. Sequence analysis The common theme among the projects of mRNA difference analysis will be supported by three key components of the Technical Core. The Affymetrix GeneChip analysis system allows the assessment of expression levels for thousands of different mouse genes. At present, 6,500 mouse genes are available on four chips. These genes are present as microarrays on a silica chip and are synthesized in situ using standard DNA synthesis chemistry as well as masking technology utilized in computer chip manufacturing. The arrays are hybridized with fluorescently labeled cDNA, using the mRNA of interest as a template, and the hybridization is """"""""read"""""""" by a scanning laser. The Affymetrix system consists of five components: the GeneChip Probe Array, GeneChip Fluidics Station 400, Hewlett-Packard GeneArray Scanner, and GeneChip Probe Array, Gene Chip Software. Together, this system has automated many steps in the hybridization, washing, and data capture and analysis.
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