Instruments Available for Structure Determination
Bruker AXS X8 Prospector Ultra Apex II CCD Kappa Goniostat, Quazar MX optics and Microfocus Source and Oxford 700-series cryosystem.
Bruker AXS X8 Prospector Ultra microfocus IµS sealed-tube X-ray generator featuring a highly sensitive charge coupled device (CCD) APEX II detector for rapid data acquisition and crystal analysis (MC 1-6811). The X-ray facility was recently upgraded in 2010 by an NIH/NCRR shared instrument grant (1S10 RR026501) worth $477,916 awarded to Dr. Joseph Wedekind.
The state-of-the-art system from Bruker AXS comprises an I(μ)S microfocus sealed tube Cu(Kα) X-ray source, integrated optics, and a highly sensitive APEX II CCD detector that can read out diffraction images in ˜1 sec. An Oxford 700-series cold stream is available to cool crystals to 100 K for data collection.
Close up of X8 Prospector I(μ)S microfocus sealed tube Cu(Kα) X-ray source, integrated optics, and APEX II CCD detector.
In addition, the upgraded system includes a
point-and-clickmotorized goniometer head for rapid sample centering. The CCD detector is mounted on a 4-circle platform to rapidly obtain the most complete data sets. The 0.120 mm beam size produced by Bruker's multilayer Quazar optic is well suited for small crystals. The system can resolve a 295 Angstrom unit cell for challenging applications. A Mosquito liquid-handling robot (TTP LabTech) is available for rapid setup of nanoliter-scale crystallization experiments.
Nearby temperature-controlled rooms equipped with microscopes are available for crystal growth, documentation and mounting. The associated computational and stereographics facility houses several Linux and Mac workstations. Graphics and computational packages include: Coot, O, PyMol, CNS, CCP4, Phenix, SOLVE/RESOLVE, and HKL2000. The X-ray facility is located in the Department of Biochemistry & Biophysics. The executive director is Dr. Joseph Wedekind.
- Mosquito crystallization robot (TTP LabTech) for high-throughput, nanoliter-scale crystallization experiments (MC 1-6824).
- Microscopes for crystal viewing (MC 1-6807).
- Computers and software for structure determination and graphics (MC 1-6818).
- Dynamic Light Scattering (DLS) DynaPro Plate Reader II (Wyatt Technologies). Automated DynaPro Plate Reader II with onboard Domestic camera for industry standard microwell plates (96, 384, or 1536 well plates), fully integrated Dynamics software, and Peltier-driven temperature regulation that permits control of the plate from 4C - 85C with an ambient temperature of 24C. Dry gas required for operation below 20C. (MC 3-7418).
Instrument Available for Molecular Interaction Analysis
- BIAcore T200 for automated surface plasmon resonance (SPR) to attain high-quality kinetic or affinity data to quantify molecular binding interactions. Instrument sensitivity allows detection of macromolecular interactions, as well as macromolecular interactions with small molecules (>100 Da) (MC 3-8536). The BIAcore T200 purchase was funded by an NIH/NCRR shared instrument grant (1S10 RR027241) worth $386,596 awarded to Dr. Clara Kielkopf.
- VP-ITC (Microcal) isothermal titration calorimeter to determine the affinities and thermodynamic basis for macromolecular interactions.
- Fluoromax-3 fluorimeter to study macromolecular interactions using fluorescence.
Instrument Available for Determination of Molecular or Particle Size
Wyatt technology’s DynaPro Plate Reader II instrument allows for high-throughput data collection of Dynamic Light Scattering (DLS, a.k.a. Quasi-elastic Light Scattering) data which can be used to determine the hydrodynamic radius (size) and size distribution of polymers and biopolymers in solution.
- Determine the hydrodynamic radius (Rh) and sample polydispersity (Survey multiple buffer conditions to find the buffer that gives the lowest %polydispersity)
- Determine the Critical Micelle Concentration (CMC) of surfactants under a variety of sample conditions (pH, temp, etc.)
- Plate reader accepts 96, 384, 1536-well glass bottom plates with approximate minimum sample volumes of 100, 50, and 5 µL respectively (Average read time for a full 384-well plate is <1 hour)
- Temperature controlled operation allows for the determination of polydispersity, size, and thermal stability between the range of 4- 85°C
- Onboard camera allows user to capture images of each well so images can be inspected for precipitant, bubbles, etc., which can lead to poor results
- The latest DYNAMICS software is available to collect, display, and analyze your data
The minimum concentration of the material under investigation depends on the molecular weight, the larger the polymer the lower the conc. that can be sampled. Using lysozyme (14 kDa) as an example a sample conc. as low as 0.125 mg/mL can be used whereas a protein with a molecular weight of ˜120 kDa could be used at a conc. of 0.015 mg/mL. We recommend starting with 0.5 - 1 mg/mL and trying multiple concentrations. The volume of sample needed depends on the plate used (see above) and the number of wells used (duplicate samples are recommended). Dr. Jenkins is available to help new users on the design, implementation, and analysis of DLS data on a fee-per-service basis.
Instrument Available for Purification of Proteins and Peptides
ÄKTA chromatography systems are robust user friendly systems widely used in both academic and industry labs. The ÄKTA Pure is a modular chromatography system for purification of proteins and peptides. The system is designed to allow for the easy addition of new modules as our purification needs evolve over time. The system supports a wide range of chromatography techniques and meets the automation requirements needed to deliver the highest purity. The ÄKTA’s user friendly UNICORN™ software has been designed to work with GE columns and media to meet any purification challenge.
- The Pure measures the UV absorbance at a fixed wavelength of 280 nm and is capable of a 0.001- 25 ml/min flow rate and has a max system pressure of 20 MPa
- The system accepts chromatography columns sold by GE and other companies such as Bio-Rad and maintains a library of commonly used columns to aid in purification method generation
- The ÄKTA has inlets for up to 4 buffers so it can equilibrate, elute, clean, and then prepare the column for storage without additional manual intervention
- Chromatography columns can be run in both directions to aid in cleaning and due to the unique column by-pass mode columns do not have to be removed to clean the system
- The system is equipped with a pH valve (can be taken off-line when not needed) that allows for simultaneous monitoring of pH and conductivity during such tasks as ion exchange chromatography
- The ÄKTA Pure and its fraction collector are stored in a 4 °C cold box and the fraction collector accepts 10-18 mm commonly used tubes such as Corning and Falcon 15 ml tubes
- The latest UNICORN software is available to collect, display, and analyze your data
- All knew ÄKTA users must undergo training with Dr. Jenkins on the instrument before being allowed to use the instrument alone, the amount of training depends on the users familiarity with ÄKTA systems
- Dr. Jenkins is available to help users with purification experiment design, implementation, and analysis of data on a fee-per-service basis
- The purchase of the ÄKTA Pure system was made possible through the support of the Center for AIDS Research (CFAR)