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Routine quality control on samples submitted to the HTX Facility (free of charge)

 

The identification of crystallisation conditions for biological molecules largely relies on a trial-and-error process in which a number of parameters are explored in large screening experiments. A significant effort is dedicated to sample characterization and quality-control experiments in order to identify at an early stage and prioritize those samples which would be more likely to crystallize. The Sample Preparation and Characterization facility (SPC) is performing analysis on each sample submitted to the High-Throughput crystallization facility at EMBL-Hamburg using the 3µl of sample from the left-over of the robot run. At this time, the SPC facility is performing MALDI-TOF mass spectrometer analysis and a thermal denaturation assay (Thermofluor). These methods provide additional information to the users which can be used to optimize protein stability and determine optimal temperatures for crystallization experiments. You are encouraged to consult your result to check whether the sample you submitted behaved as expected, or whether there is room for improvement. Several iterations of optimization and quality control will increase the success rate in crystallisation. The MALDI-TOF analysis and the thermofluor experiments are usually done every Friday and will be available through CRIMS the following week.
To access this data you have to login and then click on menu User and sub-menu Thermofluor Experiments. The table shows all your crystallization runs done at our platform. The icon representing tubes gives you access to the thermofluor graphs. One tube brings you to a single graph whereas a batch of tubes will show the graphs of the complete trial in one figure. If you do not have icons it means your results are not available. In the one-sample graph mode you can recalculate the TM by clicking on the highest and lowest point of the graph and then clicking on the TM button. If you are interested in having your old samples thermofluor graphs registered please send an email to us naming your login, sample name and run date of the experiment. If you have any questions regarding the report please contact us: .


Thermofluor assay - Target proteins used for crystallization can be studied by thermal denaturation assays to characterize protein stability. The protein of interest is incubated with a fluorescent dye (Sypro) to monitor protein thermal unfolding: this dye becomes highly fluorescent when it binds hydrophobic protein regions. The protein is gradually heated, in order to slowly unfold, exposing these hydrophobic patches. The fluorescence signal is then used to determine the protein melting point (Tm). It is known empirically that the more stable a given protein is (higher Tm) the more likely it is to crystallize. Please note that the Tm value is influenced not only by the nature of the protein itself but also by its chemical environment (e.g., pH, salt nature and concentration, additives, presence of natural or synthetic ligands, etc.). Users interested in optimizing their sample by screening stabilizing conditions such as different buffer systems and/or additives may contact the SPC Facility for further information. Optimization is especially recommended when the Tm is below 45 degrees.


MALDI-TOF (Matrix Assisted Laser Desorption and Ionisation - Time Of Flight) is a high sensitivity and high precision technique for measuring the molecular weight of a range of biochemical molecules. For most compounds, the ionisation process does not degrade the sample so the mass of the intact molecule can be measured. The instrument provides a rapid and easy way to characterise peptides, proteins (up to 200kDa) and other biological polymers. In addition to the MW determination, the MALDI-TOF can be used for standard quality control assessment since this instrument permits measurement of complex mixture (impurity, degradation product, etc.). However, the method does not provide quantitative data. UNFORTUNATELY, NOT AVAILABLE AT THE MOMENT!


Samples characterization and optimization available upon request at the SPC Facility

 

Mass Spectroscopy services
  • Protein assessment and MW determination.
  • Selenomethionine incorporation verification.
  • Crystal content verification.
  • Peptide mass fingerprinting.
  • Identification of stable domains by limited proteolysis.
Thermal Denaturation Assay Services
  • Rubic Screen (Molecual Dimentions, 96 conditions).
  • Additives screen (96 conditions - optimized for purification and crystallization).
NEW - Boivin S, Kozak S, Meijers R. (2013) Optimization of protein purification and characterization using Thermofluor screens. Protein Expr Purif. 91;192–206.
Circular Dichorism Services
  • CD spectrum and secondary structure analysis.
  • Thermal denaturation assay.
Other services
  • AKTA purifier system.
  • Dynamic light scattering.
  • SDS-PAGE and Westernblot.
  • Isothermal calorimetry (ITC).
  • MST and nDSF
  • SPR

For more information and pricing contact us at