Techniques
Our laboratory uses a variety of methods that span the range from structural biology and biophysics to cell biology and in vivo studies.
STRUCTURAL and BIOPHYSICAL METHODS
Crystallography: Data collection is done at the synchrotrons, particularly those in the bay area (SSRL, ALS). Crystallography of receptors is all done in collaboration with the PSI GPCR Network.
NMR: We have access to a 600 MHZ NMR equipped with a TCI cryoprobe, in the basement of the pharmacy building. In addition, the Chem-Biochem NMR facility has 500, 600 and 800 MHz NMRs.
Surface Plasmon Resonance: From measuring chemokine:receptor, chemokine:GAG and other protein:proteins interactions there are several instruments on campus.
Circular Dichroism: We have access to a Jasco J-815 CD with a fluorescence accessory that allows simultaneous or non-simultaneous detection of fluorescence and CD. It also has an automated dual syringe titration apparatus and Peltier temperature control.
Mass Spectrometry: , We have access to a 7 Tesla Thermo Finnigan LTQ/FTICR MS instrument (Dorrestein Lab) and a Bruker Daltonics Microflex MALDI-TOF system for protein and peptide identification and proteomics work (Pharmacy School instrumentation). We also have access to a Thermo Finnigan Orbitrap (Susan Taylor Laboratory). There is also an excellent biomolecular/proteomics mass spectrometry facility in the Chem/Biochem Department.
BIOCHEMISTRY and CELL BIOLOGY
Protein Expression: Our work depends heavily on recombinant (bacteria, insect cell and mammalian cell) protein expression and purification, for which we have two Akta FPLCs, and two HPLCs. We also have a Nanodrop for measuring protein and nucleic acid concentrations using 1ul samples. It is a must have! We also have a tissue culture facility for mammalian and insect cell expression, and for cell biolog.
BRET: We have a Perkin Elmer Victor Light Luminescence Counter with liquid handling capabilities for conducting Bioluminescence Energy Transfer (BRET) experiments for looking at protein-protein interactions in live cells.
Radioactive SPA Binding Assays: A Perkin Elmer Microbeta Trilux Microplate Scintillation and Luminescence Counter is used for radioactive SPA binding assays to measure chemokine:receptor affinities and for competition binding assays.Plate Readers: These include a Molecular Devices Flex Station which has a plate reader format with liquid handling capabilities for kinetic analysis of biochemical and cell based assays like Calcium Flux and BRET assays. It can do absorbance, fluorescence, fluorescence polarization, luminescence and time resolved fluorescence, and transfer materials from one 96 well plate to another. It has a dual monochromator for ratiometric and multiplex applications. We also have SpectraMax M5 (Molecular Devices) plate reader that has capabilities for fluorescence, fluorescence polarization, time resolved fluorescence, absorbance, and luminescence. It is a dual-monochromator, multidetection plate reader that can accept 96-384 format plates.
Flow Cytometry: We have a Guava EasyCyte 8HT which is effectively a benchtop microcapillary flow cytometer for use in individual laboratories. It can be used for cell surface protein expression quantification, various signaling assays like cell cycle, apoptosis etc. It is the top of the line model with 6 colors, back and forward scatter, accepts 96 well plate in addition to tubes.Automated Cell Viability and Counting: A Beckman Coulter Vi-Cell allows for automated cell counting and viability measurents.
Calit2/Other: The Nano3 facility at UCSD supports the use of high tech equipment for UCSD faculty and students, all with user training. Other relevant core facilities include the imaging/microscopy center and FACS core in the Cancer Center.