|
|
|
|
| |
Technical Support > FAQ'S |
 |
|
| |
|
|
| |
1 -. I grew crystals of my protein by the hanging drop method and I would like to improve their quality. The crystallization conditions I used are the following:
In the drop: 6 mg/ml protein and 12% ammonium sulfate;
In the well: 24 mg/ml ammonium sulfate.
How can I extrapolate these conditions to the counter-diffusion technique?
2 -. It is possible to use the GCB for screening of crystallization conditions?
3 -. I wonder how GCB can be used for micro-gravity experiments?
1 -. I guess you have your protein at 12 mg/ml concentration and then, when you mix equal volumes of the protein solution and the (NH4)2(SO4) you got the actual concentration in the drop. Let us plot your drop evolution in the phase diagram. The starting situation in the drop in red is C0. The starting concentration in the well, in blue is unlabeled. The concentration in the drop changes because it evaporates. Therefore, as the water molecules move from the drop towards the well the concentration changes along the dash line starting in the origin of the plot and passing through the starting conditions of the drop. If the well drop system is completely closed, the evaporation of the drop is driven by the difference in vapor pressure in the drop and in the well. Therefore, the maximum achievable concentration of protein in the drop is Cmax, obviously twice the starting protein concentration. If you got crystals in the drop, certainly that maximum concentration was never achieved. Also, if you got crystals in the drop it means that the location of the super-solubility curve is between C0 and Cmax.
You can use your starting protein and precipitating agent solution (those you use to make the drop). In your particular case, you should fill the capillary with your buffered protein at concentration 12 mg/ml. Then prepare the gel of agarose with the same buffer. Finally, punch the capillary into the gel and pour onto the gel layer the ammonium sulfate solution at 24%.
back to top
2 -. Yes, it is. I'm working on optimizing the screening capability of the counter-diffusion techniques but I have not yet a complete protocol for it. My suggestion, for now, is the following one:
- Use 0.1 mm capillary to minimize the volume of protein.
- Select your preferred screening (Hampton Screening, etc)
- Put each one of the chemical cocktail in the GCB.
- Fill the 0.1 mm capillary with the protein solution at a reasonable concentration (10 to 20 mg/ml).
- Put the capillary in the guide and be sure that the lower end is soaked by the chemical cocktail.
- Note that you can use the same cocktail for six different proteins (three if you test two protein concentrations.
- If nothing happens after two days, remove the chemical cocktail and replace by the same one at higher concentration.
back to top
3 -. GCB can be used for micro-gravity experiments as a passive reactor, without any moving part or crew manipulation. The method is simple:
To perform micro-gravity experiments one always has to supply the reactors a few hours before the launch time. Let us call this time the waiting time for launching. After take-off, there is a time required to reach the orbit around the Earth and (for the case of the International Space Station) to be locked to the ISS and to locate the reactor in the proper place. Let us call this time the waiting time for orbiting.
Taking into account the above considerations, you prepare the GCB and punch the capillaries into the gel with a depth x such that:
x > (D· t) 1/2
where D is the diffusion coefficient of the precipitating agent and the waiting time for launching plus the waiting time for orbit. As a result, during the waiting times for launching and orbiting, the Earth, the precipitating agent will reach the protein solution filling the capillaries and the crystallization will take place in gel-free solution under diffusion controlled mass transport.
Because of the restrictions of volume and mass in the space orbiter, one of the major drawbacks to crystallize macromolecules in space is the reduced number of reactors which precludes screening of crystallization conditions. If we consider that it is possible to place 39 GCB (234 capillaries) in a volume of 10x10x10 cm3 with a mass of about 1 kilogram (solutions and gels included), GCB is a simple and inexpensive device to be used in space crystallization.
back to top
|
|
|
|
