On-site X-ray facility
The on-campus X-ray facility includes two X-ray diffraction systems.
- Two Rigaku RU-300 copper rotating anodes equipped with Raxis IV dual imaging plate detectors
- High Flux confocal optics (Molecular Structures Corp.)
- X-Stream cryo-cooling system
- Linux computers for data collection
LS-CAT Synchrotron Beamlines
UM is a member of LS-CAT, a consortium of academic and research institutions that operates a multi-beamline facility located at Sector 21 of Argonne National Laboratory's Advanced Photon Source. This facility consists of four experimental stations for X-ray crystallography with insertion device sources. The primary station, 21-ID-D, is fully MAD capable and is tunable from 5–20 keV. The 21-ID-F and 21-ID-G stations are at fixed energy (12.67 keV) and are suitable for selenium SAD experiments. The fourth station, 21-ID-E, is planned to be tunable for selenium and bromine MAD. Each station is equipped with state-of-the-art diffractometers, robotic sample changers, and large-area CCD detectors. Beam diameters of 5 to 50 microns are available. Additionally, mail-in and remote access services are available.
The CSB has two environmental rooms (4 °C and 20 °C) adjacent to the wet lab that are used for crystal growth. Each room is equipped with stereomicroscopes for crystal viewing.
Gryphon Crystallization Robot
The Gryphon crystallization robot dispenses drop volumes as low as 100 nanoliters. Its array of 96 glass syringes delivers up to 100 uL of precipitant simultaneously.
Protein Purification Facility
The CSB has added a low-volume ITC unit from TA Instruments (Nano ITC) to its repertoire for biophysical analysis of protein interactions. The Nano ITC measures the thermodynamics of ligand binding and reaction kinetics. The low-volume ITC substantially decreases the amount of starting material needed compared to traditional ITC units (350 uL vs. 1.8 mL) and reduces the time required to complete a titration by one-half. Injection volumes as low as 1.4 uL at 175 sec intervals can be used and heat effects as small as 50 nanojoules are detectable. Results are analyzed using the accompanying NanoAnalyze data analysis software. The unit has a gold cell, so the only reducing agent that is compatible is TCEP at <1 mM concentration. (If reducing agents are required, the center can provide access to a Hastelloy Alloy C standard size cell in a VP-ITC.) Access to the Nano-ITC is provided through contract service or user assisted use. Access to the VP-ITC is provided through contract services or training.
The CSB has incorporated two ÅKTAxpress protein purification systems each with the capability of completing a two-step purification experiment on 4 samples in tandem without staff intervention. As an example, using proteins that have well-established two-step purification protocols, such as His-affinity tag followed by size exclusion chromatography, the center would be able to purify 8 mutant forms a single target overnight. This automation allows us to lower the cost of purifying established prep proteins, while maintaining high purity and large volume size.
High-Throughput Protein Lab
Located in LSI 3238
Equipment in the HTP Lab
The bacvulovirus process has been a “black box” in that quantitation of DNAs for input and titering of virus produced have been labor intensive, time-consuming and often unreliable.Thus it is quite difficult to trouble-shoot problems of poor protein production or changes in detected expression level. Because the insect cell/baculovirus expression process typically requires 5-6 weeks, this is a large investment of time and resources without any guarantee of success.By applying new tools, such as QPCR, the process becomes more transparent; the success rate can be increased enabling high-throughput methods. Applications for QPCR in the baculovirus process include quantitation of specific DNAs in complex mixtures, development of transfection protocols with optimal efficiency, and quantitation of virus concentration in several hours as opposed to seven days for traditional titering methods. HTP Lab equipment and protocols for doing this will be available to interested LSI users by contacting HTP staff for access and training.
The Roche MagNA Pure Compact unit automates nucleic acid isolation from tissues or other natural sources. We will use this instrument to purify DNA from recombinant baculovirus. The MagNA Pure will accept eight samples at a time and deliver QPCR-quality nucleic acid template in 30 minutes.
The Roche Lightcycler 1.5 system is a real-time, quantitative PCR instrument. QPCR monitors the incorporation of fluorescent label into the PCR product or release of label from degraded detection oligos during each cycle of PCR. The incorporation of label or release of label over time is compared against standard curves and used to detect and also quantitate the amount of specific nucleic acids in a sample.
The Biomek FX liquid-handling robot is the workhorse of the High-Throughput Protein Lab. It allows rapid and precise execution of experimental steps in high-throughput cloning and expression optimization. The dual-arm Biomek has a 96-channel pipetting head for fast transfer of volumes up to 250 uL. A gripper for moving labware is integrated with the 96-channel head. The second arm of the Biomek has a pipettor with 8 individually controlled channels for transfer of volumes up to 1 mL.
Caliper Labchip 90
The Caliper Labchip 90, pictured here, is a high-throughput replacement for PAGE analysis of protein and DNA samples. The instrument is based on a microfluidic chip. Samples are introduced in a 96- or 384- well plate, and separated proteins or DNAs are detected by laser-induced fluorescence. The separation data can be displayed either as an electropherogram showing analyte peaks or formatted as a virtual gel image. The unit allows for easy switching between DNA and protein sample analysis through selection of several pre-configured assays. The sensitivity of this system is comparable to coomassie-stained gels but the labor and time involved in sample preparation and processing is considerably less.