Crystallization Lab
The crystallization laboratory component of the Biomolecular Structure Core - Norman is located in room 2750 of the Stephenson Life Sciences Research Center on the University of Oklahoma-Norman Research Campus. The purpose of the laboratory is to help researchers at the university and throughout the State of Oklahoma crystallize protein and/or nucleic acid samples of interest.
Funding for the robotics instrumentation was provided by the National Science Foundation, Award 0922269.
The BSC-Norman provides nanoliter volume crystallization screening using the Mosquito by TTP Labtech. The Mosquito uses a continuous reel of disposable positive displacement micropipette tips capable of dispensing volumes ranging from 50 nL to 1.2 µL. The use of a disposable pipette guarantees that there is no cross contamination. Currently, the facility offers a number of commercial 96-condition and 48-condition screens. Please contact the Facility Manager regarding available screens. You may supply a non-commercial screen or a commercial screen that is not available in the MCL. If using a screen not offered by the MCL, you will be responsible for filling the tray, or, at the very least, a 96-well block, to be used on the Mosquito. Please contact the Facility Manager if you have questions regarding the use of your own screens.
The minimum volume of sample needed to set up a 96-well tray is 15 µL. This will allow pipetting of 100 x (50 nL-1200 nL) drops. Optimum drop sizes and initial screens can be determined in consultation with facility personnel. It is recommended, if enough purified compound is available, to use 300 nL of sample per drop. Furthermore, when attempting to crystallize a sample for the first time, a minimum of 10 broadscreens (=1000 conditions) should be set up.
Successful trials can then be optimized in the crystallization laboratory or at the researcher’s home laboratory.
All crystal trays will be imaged at room temperature automatically by our Formulatrix Rock Imager 1000 which has UV imaging and Multi-Fluorescence Imaging (MFI) technologies and is compatible with LCP crystallization technique plates. Crystallization trays can also be stored and imaged at 4ºC with our Rigaku Americas Desktop Minstrel with Gallery 160 imaging system.
The BSC-Norman can also provide guidance and materials to users for setting up crystallization trials manually. It is recommended that only 24-well or 48-well trays be done in this manner and all 96-well trays be set up using the Mosquito.
Upon the successful crystallization of the macromolecule of interest the laboratory will provide guidance in screening of the crystals for diffraction and subsequent structure solution.
Applicable fees will be charged for services.
Available consumables include:
- 96-well and 24-well crystallization trays
- crystallization reagents
- materials for sealing tray wells
- additives for screening and optimization
Equipment provided for conducting crystallization trials:
- pipettors and other needed glassware
- incubators for temperature control
- microscopes for visualization and recording of trial wells
Sample Purity
Samples submitted for crystallization trials should be as homogeneous and pure as possible in order to have the highest probability for success. All biochemical and biophysical information that can be obtained can aid in the prediction of successful crystallization. At the very minimum an overloaded SDS-PAGE gel should show a single band at the correct molecular weight for the compound. Other tests that can aid in determining purity and homogeneity:
- Size Exclusion Chromatography (SEC)
- Mass Spectrometry
- Isoelectric Focusing (IEF) gels
- Amino Acid Analysis
- N-terminal Sequencing
- Native-PAGE
- Ultracentrifugation
- Dynamic Light Scattering (DLS)
Sample Concentration
The other major physical property to consider for the sample is concentration. A general rule of thumb is a sample concentration of 10 to 20 mg/mL. If the sample can be concentrated to a much higher level, then it is recommended to do so, and, conversely, if the sample cannot be concentrated to that level then it should be concentrated to a level just under where the sample will aggregate or precipitate.
