I've set up some screening trays with my protein and got crystals after two days. After shooting this crystals at the synchroton, I am sure that these crystals are protein crystals. I've set up my tray with 8 mg/ml concentration of protein, fresh from purification. I've used 1ul of protein with 1ul of mother liquor.
When I set up my optimisation trays, varying only the precipitant concentration with the same concentration of protein, after two days, all my wells are filled with precipitate. In these trays, I've used 2ul protein + 2ul mother liquor and 5ul protein + 5ul of mother liquor to see if I can get any crystals growing in different dynamics. I tried growing the same protein with the same conditions at a lower concentration and after 2 weeks, nothing is growing.
I am also growing them at the same temperature.
Could there be any reason why I can't regrow my crystals/get crystals even in the same conditions?
Hi Liyana,
This is the case of troubleshooting. The best would be to eliminate one by one unknown parameters. We had a case, when just switching the plates caused us a problem. It turned out that plates were made from different plastic. First, I would recommend – to set your original plates with the original precipitant and protein. To choose the same volumes, concentrations and ratio for protein and precipitant. Second, check if freezing and then thawing of your protein effects precipitation. As it looks from your message, lowering the concentration of the protein did not resulted in any nucleation. Which means that the original concentration was OK.
If you are doing everything exactly as the first time when you had crystals, then only a reason would be a protein. It has to be fresh. You can add an extra step and run an additional purification and separate degraded protein from native.
Good luck!
If nothing mentioned above works, I suggest you also check your solutions, stocks and chemicals used for the optimised conditions. I guess you prepare the stocks manually, so check the quantities and volumes you use. It would be a good idea to use the same suppliers as the ones used for the initial condition. Also, you don't mention what lower concentration of protein you used. If the drops are clear at this concentration and you get only precipitation at the higher concentration you used, you should definitely try intermediate protein concentrations. Although you should get reproducible crystals at the initial precipitant and protein concentration. My feeling is that the problem is either your optimised solutions or the protein batch. Therefore, try different purification batches using the same crystallization conditions and see if there are any differences between batches. The purification should be performed in the same manner each time if you want to reproduce the crystals. After you reproduce the first crystals, you may try to improve the purification if nothing else works to get better crystals. Good luck!
Thank you for your suggestions! These have definitely made me more optimistic now. I'll try all of these advice and see how far I can get with it :)
Liyana,
There are additional comments.
When you freeze your protein solution, you have to make sure that this process was done fast. We usually freeze in liquid nitrogen. We divide protein solution into small volumes. In our case 120-130 uL in the single tube. That is an amount to set a single crystallization tray. We drop tubes in liquid nitrogen. Sometimes caps will popup, that is OK. And then we move tubes in a freezer. The reverse process (thaw) should be done fast as well. Our technicians warm tubes in their hand, gently mixing the solution. We found that, if we place our frozen protein solution in ice for thawing, in most cases we are getting some precipitation (we are loosing part of protein). Dividing your protein solution into small portions avoids you to freeze and thaw your proteins multiple times. You do this just ones. You can choose convenient for you volume of solution, which would make for example one crystallization tray.
When you are optimizing your crystallization conditions, you either change the volume of the drop, or prepare your own solutions. We found that both this options do not guarantee reproducible conditions for crystal grow. In small drop you can get higher saturation and thus better crystal nucleation. In bigger drops processes are running slower and conditions maybe not favorable for nucleation. In the case when you prepare your own solution, the problem is that even using solution from the same company will not match solution in the crystallization screen.
We grow our crystals in 96 well trays. 2uL drops ( 1+1, protein solution+crystal screen solution) produce dissent size crystals to collect data using the synchrotron radiation source.
I hope all these comments will help you better control your crystallization experiments and give you higher chances to reproduce your crystals.
Good luck!
Every time you set up new trays you should repeat the conditions that gave you the greatest success so far. So if 1 µl + 1 µl gave you crystals then you should repeat that and also try different drop sizes (or maybe vary the drop ratios so 0.8 µl + 1.2 µl and 0.8 µl + 1.2 µl).
If you don't get any success in your conditions identical to the previous setup then obviously there could be a problem with your protein or solutions (well, as identical as possible). This is called a positive control.
The answers from George and Diana are really good though and I don't have much to add =) George is right that freezing is tricky and some proteins will only crystallize well if purified fresh just before concentration and tray setup. Sometimes you can get away with freezing harvested bacteria so you can do the cell stuff less often and just purify a lot.
But you got protein crystals! Excellent work! Just keep at it and you'll get the structure.
- Badges
- Science method