The PCL is located in the Chemistry Building, Rm 105 on the University of Oklahoma's Norman Campus.
How it works: [See Schedule of Fees for associated costs]
User contacts the PCL Manager and discusses the protein sample he or she wishes to submit to the laboratory. The User will then fill out the Sample Submission Form and attach a hard copy to the sample. As noted below, samples are to be shipped on wet ice to the PCL whenever possible.
The crystallization scientist at the PCL will log in the sample, assign it a unique ID number for internal tracking purposes and set up the requested experiments based on the information supplied by the PCL Manager and the User.
Any sample unused by the initial experiment set-up will be aliquotted into sample sizes large enough to do individual follow up trays and then stored either at 4°C or -20°C. Each aliquot will be individually identified by barcode ID relative to the sample's internal ID number. If the samples are to be stored at -20°C, the samples will be snap frozen in liquid nitrogen (LN2) prior to placing in the freezer.
As per PCL observation protocols, each experiment (crystallization) tray will be observed on the following schedule: +1 day, +3 days, +7 days, +14 days and +28 days post set-up. Images of potential crystalline material will be taken by the PIXERA camera mounted on the microscope. An electronic copy of all images will be sent to the User with the final report using CD format.
After crystalline material is observed, each possible crystal hit will be ranked by crystal quality and total number of crystals/drop. The highest ranked hit will be used to set up one round of investigation around the conditions of the original crystalline hit.
The User will be contacted via email with a progress report to determine the interest in sending the potential crystalline hit to the PXL for preliminary crystal analysis.
An example of a completed sample submission form can be found here.
There are many factors or variables that influence the crystallization of protein from solution. A short list of the most common variables can be found here (variable_list). The production of reproducible, diffraction quality crystals is the rate-limiting step in Structural Biology. The crystallization scientist must control as many of the variables as possible in order to correctly interpret experimental data and use that information to generate further rounds of optimization. All information that can be supplied by the molecular biologist, protein biochemist and geneticist with regard to the protein's known function, assay information and purification data is critical to the success of any crystallization experiment. We do not promise that every protein will crystallize, but we do believe that lack of data can severly reduce the potential for any crystallization attempt to succeed.
Samples submitted to the PCL should be of the best quality and highest purity available in order to maximize opportunity for success during screening experiements. In order to verify the purity and monodispersity of a sample, an overloaded SDS-PAGE gel showing a single band at the correct molecular weight must be submitted. This is not the best way to determine sample purity, but it is a widely available tool that most, if not all research laboratories have access to. If available, one or more of the following tests should be reported also: Biological or Biochemical Assay (BBA) information (especially useful in tracking lot to lot variation) Amino Acid Analysis (AAA), N-terminal sequencing, Mass Spectroscopy (choose from: MS, LC-MS, HPLC-MS, MOLDI-TOF), Dynamic Light Scattering (DLS), Native-PAGE, Isoelectric Focusing (IEF) gels or Size Exclusion Chromatography (SEC). Copies of the relevant data demonstrating the purity of your sample should be enclosed with the sample during shipping.
If your sample has an activity assay, each sample sent to the PCL should have the results from a recently completed assay for the sample normalized against a standard reference. This will be important in assessing the differences between samples sent to the PCL for crystallization.
Samples should be received on wet ice (4°C) shipped overnight to the PCL, unless other arrangements have been made. For samples sent on wet ice, the sample must be in a buffer system that maintains sample stability for at least 48 hours at 4°C in order to preserve your sample during shipping. A small volume of the protein stabilization buffer used during transport (<10mL) should also be included. The PCL accepts no responsibility for samples that arrive in an unstable (aggregated or precipitated) state.
Samples that have been frozen in the home laboratory can be shipped on dry ice in frozen aliquots provided that the tubes are individually and permanently marked. A small volume of the protein stabilization buffer used to freeze the sample in (<10mL) should also be included.
In all cases, all information about the protein that you can supply will greatly assist in the crystallization of your target molecule. Knowledge about your target molecule prior to crystallization attempt is our best tool in designing experiments with the greatest chance of success.