CCR Dedicated Cores

Division Of Basic Sciences - Nci

Investigators

Abstract

Center for Molecular Microscopy (CMM) is an interdisciplinary research collaboratory, located primarily at the ATRF, specifically aimed at applying recent cutting-edge advances in the field of 3D electron microscopy to important problems within the CCR portfolio. The research program of CMM includes the development and application of novel methods for cellular imaging using correlative light and focused ion beam scanning electron microscopy (FIB-SEM), and molecular imaging using cryo-electron microscopy and cryo-electron tomography. Genomics Technologies Lab (GTL) provides services to CCR investigators in microarrays, Whole Exome capture and sequencing, Nanostring gene expression analysis, and other specialized nucleic acid detection technologies. The Protein Expression Lab (PEL) provides dedicated support to CCR investigators for cloning of expression constructs, protein expression, and protein production. Cloning services includes the construction of expression plasmids for downstream protein, shRNA and CRISP/CAS9 expression. For microbial and eukaryotic expression, transient and stable expression of proteins in a variety of systems including, E.coli, yeast, insect, mammalian cells, or cell-free expression is provided. The lab provides parallel small-scale screening in bacteria, yeast, insect, and mammalian cells to optimize protein quality using appropriate platforms to test multiple expression modalities side by side to determine for each target protein the most suitable method for purification. PEL also provides scaled-up production for tens of liters of culture and hundreds of milligrams of protein. Custom recombinant lentivirus production to study transgene or shRNA delivery for gene knockdowns in vitro and in vivo is also offered and includes virus concentration and titration services. The Protein Characterization Lab (PCL) provides dedicated support to CCR investigators for protein quantitation, identification and characterization, including protein interaction partners isolated from mammalian cells. Protein Identification is performed from SDS PAGE gels, co-immunoprecipitation and related methods. Qualitative and quantitative analyses using high resolution, accurate mass data for high confidence identifications and good quantitative reproducibility is provided using a variety of mass spectrometry techniques. The lab has capability to identify posttranslational protein modifications such as glycosylation and phosphorylation using mass spectrometry methods. PCL also has the expertise in quantitation by isotopic labeling of cells in culture (SILAC) or by reaction with isobaric tags (iTRAQ/TMT) for analysis of proteins from mouse or clinical samples. Quantitation can be multiplexed, allowing comparison of multiple conditions/states/treatments at once. They can also perform label-free relative quantitation and absolute quantitation of specific proteins in a complex mixture. In a targeted approach, they can perform absolute quantitation of the level of a particular protein in a complex mixture (e.g., serum, conditioned media, lysate). PCL also provides targeted and global metabolite analysis. The Optical Microscopy and Analysis Laboratory (OMAL) provides, develops and applies technologies for quantitatively understanding the molecular basis of cancer and HIV infection in the spatial-temporal context of the cell and tissue. The available technologies range from standard fluorescence microscopy to super-resolution and intra-vital two-photon imaging, and computational capabilities for visualization and analysis, including multiplex staining of tissues and cells including the Akoya CODEX technology. The Single Cell Analysis Facility on the Bethesda main campus provides the latest technologies for CCR-investigators to interrogate gene expression, the immune repertoire, and epigenetics of hundreds to thousands of individual cells. These methods allow for revealing the true complexities of cancer with sharper resolution into the cell types and mutations that drive cancer. Since the pandemic began, the cores have supported several projects working on COVID-19 research and vaccine development.