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The following is a list of the most frequently asked questions;
Whom do I contact? - See our contact page and get in contact with either Mike or Jens. How pure does the sample need to be? - An important criteria is the purity and homogeneity (or monodispersity) of the protein solution. The protein should be as pure as possible with certainly no more than 10% impurities. An estimate of purity using a gell, DLS or other assy method is encouraged. Any impurity greatly reduces your chance at successful crystallization. What is the optimum concentration for my sample? - Typically 10mg/ml is a good goal to aim for but there is nothing magical about this figure. Sometimes a protein will start the precipitate at lower concentrations; this is not good. Other times a protein is quite soluble at 30mg/ml so a concentration high than 10mg/ml is advisable. The bottom line is that it depends on the solubility of your protein. Remember that at crystallization you are trying to 'nudge' your solution into a supersaturated state that will produce crystals. How much protein do I need? - The simple answer is as much as possible but of course there is more to it than that. There are essentially three options which require differing amounts of protein. Manual Set-up: Each crystallization trial (or well) will use 2 ul of protein solution. A typical set-up would have 96 wells for each of 10-15 different screens; each screen would require 192 ul of protein solution. Mosquito Robotic Set-up: Each well is typically 0.1 ul of protein solution. Therefore, one 96 well screen will require approximately 10 ul of protein solution. Fluidigm Nano Set-up: Total amount of protein solution required is 1.4 ul for a 96 well screen. Each option can be set-up at room temperature and/or low temperature. Is there a rational place to start trials? - A usefull place to start may be to check if similar proteins have been crystallized and if so under what conditions. There are searchable databanks where this can be accomplished. If there is no useful information available then a full screen of ~1500 conditions (96 wells, 15 screens) should be tried. Where do I set up crystallization trials? - You can attempt to crystallize your protein in your own lab but the most preferable would be in BI114 or the Broad cold room where each room is set up specifically for crystallization trials. Where do I get the supplies needed? - The personnel in the Molecular Observatory will assist you with identifying appropriate supplies and vendors, then you may purchase supplies as is usual for your research group. Who will collect the data? - The personnel in the Molecular Observatory will take the lead in collecting and reducing data but it is expected that the investigator will participate as an interested observer towards the end of being able to assume most of the responsibility if numerous data collections are undertaken. Who will solve and refine the structure? - The personnel in the Molecular Observatory will render all necessary assistance in the solution and refinement of your structure with the expectation that you will gain near independence as the work progresses. How much will it cost? - Good question. It depends. Contact Mike for details. |
