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Protein Localizattion, Identification and Folding Core
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In Vivo Animal and Human Studies Core

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Protein Localization, Identification and Folding Core

Facilities and Resources

Overview

The administrative and part of the services home of the PLIF Core are located in the Department of Molecular and Integrative Physiology on the 7th floor of Med Science II building (Med Sci II) and in the Pathology Department located in the 4th floor Med Science I (which is connected with the Med Sci II). Core recharges will be managed by the Center Administrative Core (Dr. Jeff Cole).

Equipment is present in a procedure room for fixing and embedding frozen cells and tissues and embedding in resin for semi-thin and thin sections for EM.  A Leica 1950 Cryostat is present and maintained by Mr. Bradley Nelson. Ten feet of desk space and a carrel for the Core microscopy technician is present in the procedure room.   A Leica Laser Capture Dissecting Microscope is also located in an adjacent small room.  In immediate proximity there is also dedicated space and equipment is present for carrying out immunohistochemistry.  Microscopes are present for preliminary examination of sections and a Nikon inverted scope equipped for fluorescence microscopy with a digital camera is maintained by the Core. There is also refrigerator and freezer space to store Core materials. Both Drs. Williams and Omary have offices across the hallway from this dedicated space. All core personnel have networked desk and laptop computers. Two conference rooms are also adjacent to the laboratory rooms.


The Morphology and Image Analysis Laboratory (MIL) which contains microscopes used by the Core, is a medical school core facility and is located on the A level (basement) of the Biomedical Science Research Building (one block from the Medical Sciences Buildings). It is equipped with a Phillips CM-100 transmission electron microscope (EM) equipped with a computer controlled compustage and a high resolution (2K X 2K) digital camera. AMT software drives the digital camera and provides on screen live viewing. Scale bars are automatically created for incorporation into saved images as a separate operator function if desired. The MIL contains 5 confocal microscopes including a Olympus Fluoview 500, a Zeis LSM 510-META mounted on an inverted Zeiss microscope (Using the META system, the researcher can easily separate highly overlapping emission peaks), a Nikon A1 confocal with diode based laser system, and two Leica SP5X multiphoton confocals, one with FLIM on a Leica inverted microscope and the other on a upright microscope. In addition there are three high quality microscopes equipped for fluorescence and Nomarski optics and high resolution digital cameras and a Deltavision-RT Live Cell Imaging System with programmable stage. All of these instruments plus workstations for image analysis are available after mandatory training for use by GI Center investigators. Recharge fees are set to cover the costs of maintenance and staff time. The MIL also contains three Reichert ultracut-E ultramicrotomes, two Leica rotary paraffin microtomes and a Tissue-Tek paraffin embedding work station. A permanent staff maintains the instruments and provides instruction in their use.


The Aperio AT2 scanner is located in the Unit for Laboratory Animal Medicine (ULAM) Core Facility at the North Campus Research Complex of the University of Michigan (15 minutes door to door shuttle service). ULAM has staff who will pick up the slides from the shuttle or from the medical campus to make their services as user friendly as possible.


The other microscope core used by GI Center members is the Morphology and Image Analysis Core (MIAC) of the Michigan Diabetes Research Center. It is located in the Brehm Diabetes Center, four blocks from the Medical Science Division (10 minute walk). This building also contains the Kellogg Eye Center. This Core contains four confocal microscopes all equipped for four color imaging. An Olympus Fluoview 500 is used for routine confocal analysis. A Nikon A1 confocal is designed for evaluation of both fixed and live cells.  It is based on a Nikon inverted Ti-E microscope with Nikon’s perfect focus system, a Prior motorized stage for tiling large areas and multi-point imaging, and a Tokai Hit environmental chamber that regulates temperature and CO2 which has been modified to fit on the Prior stage. The third microscope is a Leica TCS SP5 confocal shared with Ophthalmology. It has a AOBS Acousto Optical Beam Splitter which allows excitation at up to 8 lines simultaneously and 4 spectral PMT detectors. It also has a motorized stage and accepts the Tokai Hit environmental chamber. The Leica application suite has integrated programing for specialized techniques like FRET, FRAP and photoactivation. The fourth system is a multiphoton Leica SP5 available through arrangement with Ophthalmology. In addition the Core includes a multi-wavelength wide field imaging system for live cell fluorescent probe imaging and several high end computer workstations for off line image processing and 3D rendering with image analysis software. This facility is maintained by a full time lab director, Stephen Lentz, Ph.D. who also provides instruction and advice to all users. The MIAC is overseen by Dr. John Williams as Core Director.


The Proteomics Resource Facility (PRF) is part of the Protein Folding Disease Initiative and is located within the Department of Pathology at University of Michigan on the fourth floor of Medical Science I building (with walkways connecting this building to Med Science Building II where the fixing and embedding of frozen cells and tissues and embedding and sectioning for EM is carried out). It occupies 1500 sq. ft. of space and is directed by Dr. Alexey Nesvizhskii. It is equipped with two state of the art Thermo Scientific mass spectrometers devoted exclusively to proteomic analysis: 1) Orbitrap Fusion Tribrid with ETD (obtained through Protein Folding Diseases Initiative funding in 2014), 2) Q Exactive HF (obtained in 2016). Management of the Core is by Venkatesha (Venky) Basrur, PhD who reviews requests together with Dr. Nesvizhskii, and advises members of the GI Center in the use of the facility. Major functions of the PRF are to: 1) Carry out qualitative identification of proteins from multiple sources such as Coomassie and MS-compatible silver-stained gels, shot-gun proteomics analysis of complex mixtures using two-dimensional liquid chromatography and mass spectrometry (2D-LC/MS), 2) Identify post-translational modifications such as phosphorylation, ubiqutination, acetylation, (mono-, di-, tri-) methylation, glycosylation etc., 3) Carry out quantitative proteomic profiling using isotope labeling (SILAC, TMT) and label-free quantification strategies, 4) To identify protein interactions and complexes using Affinity Purification Mass Spectrometry (AP-MS), and 5) Provide support in terms of experimental design, technical training (related to sample prep for MS analysis), data interpretation and bioinformatics analysis.  The PRF also provides support for Absolute Quantification (AQUA) of multiple analytes using a Parallel Reaction Monitoring (PRM) technique.


The Protein Folding Diseases (PFD) Initiative Resource Center, which provides the Antibody Reagent Library and the Human Tissue Brain Bank is located at the North Ingalls Building (room G179), with a walkway to the Medical Science buildings I and II. This is a dedicated room with two refrigerators, an ultratemperature freezer and lab bench and routine lab equipment. A technician (Mr. Matthew Perkins) oversees these aspects of the Resource Center. Other aspects of the PFD Initiative administration (e.g., disseminating information related to the PFD pilot program, seminars) are overseen by Catherine Bearman (administrative specialist with the Department of Pathology).

 

Equipment is available for fixing and embedding frozen cells and tissues, and embedding in resin for semi-thin and thin sections for EM. Core members have access to a Leica 1950 Cryostat that is maintained by Mr. Nelson. The Leica Laser Capture Dissecting Microscope (LCM) system (Leica LMD7000 microdissection system) is a new addition to the Core. These instruments are located in the Physiology Department in Medical Science II building. The LCM allows specimen collection by gravity (contact-free and contamination-free), and is being maintained by Yongjia Feng in the Omary laboratory who will also facilitate training. Additionally, the Core has space and equipment for immunohistochemistry. Microscopes are present for preliminary examination of sections and a Nikon inverted scope equipped for fluorescence microscopy with a digital camera is maintained by the Core.
     The MIL is a Medical School Core facility (https://medicine.umich.edu/medschool/research/office-research/biomedical-research-Core-facilities/microscopy-image-analysis) that is located at the Biomedical Science Research Building, one block from Medical Science II building. It is equipped with a new JEOL 1400-plus electron microscope. This system offers accelerating voltages from 40-120 KV in 20 KV steps and is equipped with two high end digital cameras for superior resolution.  The stage is equipped with high tilt specimen holder offering tomographic images which can be 3D volume rendered. Mr. Nelson uses the instrument to take pictures for those not wishing to obtain training and take their own. The MIL also houses 5 confocal microscopes including an Olympus Fluoview 500, a Zeiss LSM 510-META with a META system, a Nikon A1 confocal with diode based laser system, and two Leica SP5X multiphoton confocal microscopes, one with FLIM on a Leica inverted microscope and the other on a upright microscope. The MIL has also recently purchased a new Leica TCS SP8 two photon microscope with FLIM and FCS. To increase spectral flexibility of this new microscope, the Acousto-Optical Beam Splitter (AOBS) and white light laser (WLL) is configured to allow tuning of the system with up to 8 simultaneous excitation lines. The tunable Coherent Chameleon 2-photon laser provides deep tissue imaging and also offers spectral separation.      
     In addition the MIL contains three high quality microscopes equipped for fluorescence and Nomarski optics and high resolution digital cameras and a Deltavision-RT Live Cell Imaging System with programmable stage. All of these instruments plus workstations for image analysis are available for use by GI Center investigators after mandatory training. The MIL also offers three Reichert ultracut-E ultramicrotomes, two Leica rotary paraffin microtomes and Tissue-Tek paraffin embedding work station. A permanent staff maintains the instruments and provides instruction in their use. These amenities are easily accessible to Center Members who have not had any problems with access. 
      The other microscopy Core used by GI Center members is the Morphology and Image Analysis Core (MIAC) of the Michigan Diabetes Research Center that is located in the Brehm Diabetes Center, three blocks from the Medical Science Division. The MIAC contains four confocal microscopes that include an Olympus Fluoview 500, Nikon A1 confocal based on a Nikon inverted Ti-E microscope that is equipped with a perfect focus system, motorized stage for tiling large areas and multi-point imaging, and a Tokai Hit environmental chamber that regulates temperature and CO2. The third microscope is a Leica TCS SP5 confocal shared with Ophthalmology that has an AOBS Acousto Optical Beam Splitter which allows excitation at up to 8 lines simultaneously and 4 spectral PMT detectors and is equipped with a motorized stage and Tokai Hit environmental chamber. The Leica application suite has integrated programing for specialized techniques like FRET, FRAP and photoactivation. A multiphoton Leica SP5 is available through arrangement with the Department of Ophthalmology. In addition the MIAC includes a multi-wavelength wide field imaging system for live cell fluorescent probe imaging and several high end computer workstations for off line image processing and 3D rendering with image analysis software. This facility is maintained by a full time lab director who provides instruction and advice to all users. The MIAC is overseen by Dr. John Williams, as the Core faculty director. One important benefit for these multiple instruments is to provide proximal access to Center users and to also provide timely access without the need to wait.
     The Aperio AT2 scanner plus software is housed in and operated by the ULAM In-vivo Animal Core and will be used to generate digital images. A designated ULAM technician scans hematoxylin and eosin stained tissue sections while the users perform Aperio image analysis. Users will receive training from Drs. Robert Sigler and Nusrat. Since the scanner is high capacity (400 slides), turn-around time is within 2 days at most. Files can be saved in Mbox (a password protected internal server storage system), an external hard drive or a flashdrive as a proprietary .svs file, which can be converted to TIFF or other file formats for image analysis using Aperio toolbox software.
     The PLIF Imaging Program Core will also stock reagents for fixing tissue with formaldehyde or glutaraldehyde, labeled second-stage antibodies, fluorescent nuclear stains including DAPI and DRAQ-5 and red and green labeled phalloidin for staining filamentous actin. This task will be performed by Mr. Nelson.

Proteomics Core Program

Please be sure to submit this core services request form and submit to the appropriate Core Director to be eligible to receive the subsidies.

This facility houses 2 state-of-the-art mass spectrometers including the most recent acquisition of a high-resolution, ultra-fast and sensitive Q Exactive HF. Both mass spectrometers are used exclusively for proteomics work. Apart from the ability to generate high resolution data, the instruments provide various fragmentation methods such as CID, HCD and ETD aiding in high confidence identification of the analytes (peptides) and post-translational modifications on peptides.   Routinely these instruments can identify 3000-5000 proteins from as little as one microgram of the protein digest in as little as 2h, and ~8,000 proteins with extended sample fractionation. All instruments are in-line with their own UHPLC chromatography systems for peptide separation. The equipment includes 1) Orbitrap Fusion Tribrid with ETD which is a versatile state of the art instrument is used for most Core requests, 2) Q Exactive HF is a highly robust, high throughput, state of the art instrument that is the instrument of choice for large scale projects, 3) UPLC (liquid chromatography system coupled inline to MS), 3) 64-Core high performance compute cluster (Computational infrastructure for advanced bioinformatics analysis, and 4) Servers for data storage.
     Aside from routine Protein Identification Analysis and Quantitative Proteomics using iTRAQ, SILAC, TMT, or related techniques, the two new services featured in this component are: (i) Protein Interaction Mapping Services (AP-MS; experimental design support, sample prep technical training, consultation, data interpretation and bioinformatics analysis), and (ii) PTM Services (phosphorylation, ubiquitination and other PTMs; experimental design support, sample prep technical training, consultation, data interpretation and bioinformatics analysis)

Protein Folding Core Program 

Please be sure to submit this core services request form and submit to the appropriate Core Director to be eligible to receive the subsidies.

The center membership will have access to antibodies compiled by the PFD Resource Center (85 antibodies to various heat shock, ER, apoptosis and lysosomal proteins, among others). Additional reagents provided by the PFD Resource Center include a mouse tissue bank with mouse tissues (GI and non-GI organs) from three strains (FVB/NJ, C57BL/6J, Balbc, C3H/HeJ). In addition, the Resource Center provides access to a growing list (presently 25 independent lines) of genetic mouse lines that are maintained by PFD investigators and are now also available to UMGRC members. Another important resource is the ability of our Center members to partner with PFD investigators and apply for PFD pilot awards that are offered annually (typically in October) and are up to $50,000 per year.


In summary, all PLIF directors and associate directors are established scientists with expertise in their respective areas. The accessibility of the proposed programs includes selected services from institutional, University of Michigan Medical School Core laboratories. Benefits to Center members include subsidized user cost (proteomics, microscopy), access to reagents and tissues in the protein folding Core (at no charge) and experimental consultation with directors and manger who are experts in their respective fields. 

                                                                                                           

 

 

 

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