Protein and Nucleic Acid Analysis Core
Calvin Vary, PhD
The function of this core is to provide protein structural and functional analysis.
Automated DNA sequencing is offered on both shorter and longer instruments depending on the sample.
- DNA and RNA extraction is offered by robotic extraction and purification.
- High throughput (16-capillary) sequencing is available for appropriate projects.
- Deep sequencing/NextGen sequencing is available via interaction with the Tufts University Core facilities-so please inquire.
The facility formerly housed a QSTAR Pulsar quadrupole TOF tandem massspectrometer (qTOF, ABSciex), which in 2014 was replaced with a state-of-the-art ABSciex TripleTOF® 5600 mass spectrometer, appropriate for quantitative and informatics-based analyses including lipid andproteomics-oriented applications, respectively.
The TripleTOF® 5600 is equipped with a new OPT 5500 nanospray source III. For quantitative multiple reaction monitoring and metabolic studies, the facility also houses a quadrupole linearion trap mass spectrometer (4000QTRAP, ABSciex) fitted with a Turbo V and Nanospray III sources.
Mass spectrometers share a U_3000 RSLC nanoscale liquid chromatographic system (Dionex/Thermofischer) to enhance TOF- and TRAP-basedanalyses, and a new (2014) PEAK nitrogen gas generator, as well as a complete suite of software applications, including: ProteinPilot™, MarkerView™, MultiQuant™, and LipidView™ software application licenses.
Additional equipment includes:
- iCycler real time PCR machine (Bio-Rad)
- two model 310 DNA sequencers
- one Model 3100 DNA sequencer (ABI)
- automated RNA isolation workstations (Autogen).
All equipment is located on the ground floor or in the Vary laboratory on the second floor. Facilities for the use of radioactive materials are located on the ground floor.
For cell imaging analysis the Core also houses the Confocal Microscopy Facility, which contains a Leica confocal microscopes SP1 and SP8. The uprigh tSP1 microscope has been upgraded with an addition of a unique 442 nm laser, which greatly facilitates FRET experiments. In addition, the facility has a dedicated computer with Imaris software for 3D reconstruction of images.
In February 2014, MMCRI received a new Leica SP8 confocal microscope equipped with Acousto-Optical BeamSplitter, which delivers bright, noise-free images with minimal photodamage.This microscope includes a 405 nm laser and a Ludin chamber for live cellobservation. The existing SP1 confocal inverted and upright microscopes have been moved to a new location in the Vary laboratory (Core director) and are available for routine studies involving moderate to high levels of fluorescence, or when photodamage is not an issue.
Recent example of Mass spectrometry applications:
Favreau,A. J., Vary, C. P., Brooks, P. C., and Sathyanarayana, P. (2014) Crypticcollagen IV promotes cell migration and adhesion in myeloid leukemia. CancerMed
Young,K., Conley, B., Romero, D., Tweedie, E., O'Neill, C., Pinz, I., Brogan, L.,Lindner, V., Liaw, L., and Vary, C. P. (2012) BMP9 regulates endoglin-dependentchemokine responses in endothelial cells. Blood 120, 4263-4273
Romero,D., O’Neill, C., Terzic, A., Contois, L., Young, K., Conley, B. A., Bergan, R.C., Brooks, P. C., and Vary, C. P. H. (2011) Endoglin regulates cancer-stromalcell interactions in prostate tumors. Cancer Res 71, 3482–3493
Oligonucleotide and siRNA ordering through IDT
DNA Sequencing: (Service Request Form)
- DNA sequencing (run only)
- DNA sequencing (core does cleanup and run)
HPLC, QSTAR and 4000QTRAP: (Service Request Form)
- Nano-liquid chromatography
- Protein identification
- Protein post translational modification
- Peptide digestion, fingerprinting and sequencing
- Multiple reaction monitoring (MRM)
Service forms must be filled out completely with as much detailed information as possible. The completed form must include the signature and account number of the Principle Investigator of the lab requesting services. Completed forms should be submitted to Barbara Conley (Vary Lab).
This component of the Protein, Nucleic Acid Analysis and Cell Imaging Core is supported by the Vascular Biology Center of Biomedical Research Excellence (COBRE) Program of the National Center for Research Resources.