Hi

Since you have not observed any precipitation, I would call it a good sign as your protein prep still must be stable in given condition. Hanging drop I assume would take longer to crystallize as compared to sitting drop.

It is still difficult to point out the reason. Few more tips though:

1. Try crystallization at 20C temperature instead of 4C

2. If you have manually prepared the buffer system, try to use the one which gave you crystals earlier (if ready-made available)

3. If manually prepared buffer system, check if the water used for preparation has same conductivity as used for earlier. That might be affecting the pH.

All the best

Hi Peter, a few remarks from my own experience.

a) At 4C, equilibration with PEG-based conditions is really slow and can take several weeks. Perhaps you just have to wait longer for crystals to appear. Also note that the equilibration kinetics of manually setup hanging drops is different from the microplate-based sitting drop screens. This website https://www.douglas.co.uk/tipsntech.htm has good tips on translating from one method to the other.

b) Following up on suggestion 2 from Ameya, what I sometimes do in such situations is prepare the hanging drop using the original solution from the screen, while preparing the reservoir solution myself with variation of the precipitant concentration. That should give you at least the highest chance of reproducing your crystals.

c) Another obvious way to follow is using the existing crystals for seeding. Again, the website that I mentioned has good tips on this.

Good luck, Tjaard

Thanks Ameya and Tjaard,

These suggestions and resource are very appreciated. I will definitely look to implement these suggestions.

Cheers

Peter

Dear Peter J Cossar ,

welcome to beautiful and mysterious world of Protein Crystallization.

When you change the setup of the drop (hanging drop, sitting drop and - not very used - sandwich drop), you are changing the dynamics of the equilibrium, hence, the translation is not straightforward. You have to play around with the drop size, drop ratio and reservoir volume. We even had a case that we had to change the crystallization plates to get crystals in hanging drop.

One silly question: if you have 2.15A resolution in sitting drop why bother to optimize hanging drop?

Best regards,

Jose

I agree with Jose A. Brito , that why to bother to optimize other method. 2.15A is far better

First of all, I think the presence of precipitation is a good omen because I think the crystal is regular precipitation. However, according to your previous experiments, there is no problem with the buffer system. According to my experience, the nucleation rate of hanging drop method is slower than that of sitting drop method, but the crystals formed are larger and more regular, which is more conducive to diffraction, which may be related to the volume and pressure change of droplets. If you want to speed up nucleation, try the 18℃ condition, or inoculate the crystals from the sitting drops into the droplets of the hanging drops to promote nucleation.

Good luck with the experiment.

Hi Jose A. Brito,

I am looking to do fragment soakings. If the hanging drop does not afford crystals I will look to use a sitting drop setup.

Cheers

Peter