i have optimize different pH at pH blow 7.0 crystal was in needle cluster form at pH above 7.0 crystal has octagonal shape but it's tiny ..?
thanks in advance
Given that the crystals are small, and that there are many of them, the obvious next step is to optimise protein concentration and precipitant concentration. It might be helpful for the discussion if you could give the full crystallisation conditions, as that might well assist experts in giving advice?
thanks Nicholas, indeed i have screened protein concentration and also precipitant at 10mg/ml 8mg/ml, 5mg/ml and 3mg/ml of protein in parallel with seeding ,still tiny crystal ..
Protein crystal size is a major factor that can limit its diffraction. In many instances, We get very very tiny crystals and it's very hard to improve. To my experience, there will be serveral method to improve your crystal size very quickly.
1. If your crystal grows with a lot of small crystal, the first thing is decrease the protein concentration and the precipitant concentration to a point that the crystal can still come out but do not tends to form a lot of nucleus.
2. if you still get many tiny crystals in the minimal protein and precipitant concentration, go seeding
3. Crystal seeding is a powerful method to control the nucleus number. Crystal seeding can be classified two types: Microseeding and Macroseeding. Microseeding is planting invisible protein seed into crystallization drop with different dilution times. Here is how to prepare the microseeding seed stocks:
a. get the crystal from the crystallization drop and put into the Eppendorf tubes with 50ul well solution
b. using the hamptonresearch seeding beads and vertax for 1-2 min
c. dilute the seeding stock with 10, 100, 1000, 10000, 100000 times
4. With the seeds ready, you can use either streak seeding or direct put it into crystallization drop by pippete. The crystallization drop needs to equibrate 4 hours before seeding.
5. Using the microseeding you can control the seed number in the crystallization trial, sometimes you can get bigger crystal, but sometimes not. If the crystal still small, you may try the Macroseeding.
6. Set up the crystallization trial with different precipitant concentration and equlibrate for 4 hours. Wash the crystal seeds with well solution and put serveral seeds into the crystallization drops
7. combining the microseeding and Macroseeding, usually you can get the bigger crystals. if you don't, you can try add the additive in the crystallization buffer.
8. If you have a huge amount of protein, try big drops say 25ul protein+25ul crystallization solution.
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