The protein sample has to be pure, preferably not in phosphate buffer, and (if necessary) with minimum required salt concentration. Denaturing gel electrophoresis can be used to measure sample purity. Native gel electrophoresis, dynamic light scattering or analytical size exclusion chromatography may be performed to measure sample homogeneity.
Which is the right protein concentration? Normally large proteins should be less and small proteins more concentrated, in the range of 5-20 mg/ml. However, individual proteins behave differently, so further testing might be necessary (see Sample optimization). Alternatively, an initial screen can be indicative of the protein behaviour, based on the types of precipitates formed:
Conditions that result in denaturation of the protein should be avoided. If it remains in its native conformation, the protein and/or precipitant concentrations can simply be decreased. Optimally, about 60% of the drops should provide precipitates/crystalline material, and about 40% should remain clear.
The minimum required sample volume is 20 ul, at the desired crystallization concentration (for 96 conditions, with 100 nl protein drop volume). Different crystallant:protein ratios, as well as different drop volumes are available. Most plates can fit more than one sample. If you only have one protein, do consider multiple protein concentrations, buffer control etc.
Sometimes a few microliters are left after the screen set-up. Please let us know if you want to save it for other purposes.
Sample impurity and/or heterogeneity can prevent crystallization. Sample purification (chromatographic), sample modification (site directed mutagenesis), or sample manipulation (use of detergents, additives) should be evaluated in an effort to pursue sample homogeneity.
The Pre-Crystallisation Test (PCT) is used to determine the appropriate protein concentration for crystallisation screening. Method and reagents: Hampton Research.
Hampton Research Additive Screen: 96 additives purchased from Hampton Research will be added to the well solution at 1/10 dilution. Drop setup is to a ratio of 1:1 (Hampton Research Additive Screen).
After filtering, the protein is dispensed in 100 nl drops using SPT Labtech’s mosquito LCP® liquid handling robot.
We currently prefer to work with MRC Crystallization Plate, which is UV friendly (former Innovadyne 2D).
The plate is sealed and imaged right after setup, and stored at either room temperature or 40C.
The plate is automatically inspected routine plate schedule follows: 0, 1 d, 2 d , every week during the month and twice the following two months.
We are able to do Ultraviolet crystal imaging (Minstrel UV imager).
We will give you access to the PixRay/Icebear software to inspect and annotate your images.
After 3 months, the plates are removed from our storage. If you want to save your crystallisation plate, please let us know in time.
The LCP technique for crystallising membrane proteins. Yor membrane protein is dispensed using SPT Labtech’s mosquito LCP®. The LaminexTM UV Plastic Base 100 Micron and the LaminexTM UV Plastic cover with frame are used to dispense your sample. The Laminex set up is compatible with UV imaging.
The Helsinki Optimisation combines the promising hit conditions with 15 different salts at two pHs to provide further screening around the hit.
You can add the Hampton Additives to your favourite condition.
You can design your own custom screen and we help you with the practical details and the available stock solutions.
To ensure accuracy in pipetting and good mixing of reagents, all screens are initially prepared in deep well format. If you want to keep the rest of your custom designed screen for manual setups, please ask to save it for you.
We can seed your hits back to Random Screens http://www.douglas.co.uk/mms.htm or any custom screen of your choice.
Any crystalline protein material can be used for seeding, including fine needles, “spherulites”, microcrystals, and irregular poorly-formed crystals.
We can also increase the crystallization drop volume to 500 nl to bring the drop equilibration speed closer to manual setups.
If you choose to pipette the crystallization drops manually, we can prepare any of our current screens in deep well format.
You can also order the custom screen conditions. These can be used to optimize further the initial hits.