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Hello, typically the image program (software) has way to draw a scale (line, or arrow) on the image. But you need to calibrate that scale with the known length. For example, you can keep a transparent ruler (scale) with mm divisions visible. Zoom into that area and calibrate the software program's internal scale to match the physical scale. Once the calibration is done you can extrapolate to all Zoom levels.

Hello Bhagyasri, when one tries to crystallize a protein, we need to slowly bring it to saturation (actually meta stable state, just above its solubility limit). Due to fragile nature of proteins, we cannot simply evaporate the water which will lead to protein denaturation. So, we use precipitant solution which are typically salts, PEGs, sugars, etc. The role of the precipitant (solution) is to make the protein salt-out (come out of liquid phase to solid phase) before the precipitant salts-out (typically, the precipitants selected have very high solubility limits and hence do not salt out before the protein does). I hope this is clear. Let me know. Thanks.

Hello Mukith Miah, Typically you want to choose the size of the loop to be about the size of crystal or slightly smaller. You can bring the loop below the crystal and try to pick it up. Most of the time it will work. If the crystal is too small you can bring crystal to the edge of the drop and pick it up side ways. Occasionally, you may have a film coating the drop which is denatured protein. So you want to use different loop to clean the surface and use the correct loop to pick your crystal. Practicing using not important crystal will help you get a hang of the process. It looks hard but it is will turn out to be easy once you try few times. Let me know if answered your question properly.

@@thayumanasomasundaram699 Yes you have very well answered my question, I've practiced on some lysozyme crystals and have done pretty well after reading your comment! Hopefully I'll be able to practice on smaller crystals or needles. Thank you very much!!

Shsw Xian Au, Your concerns very valid. Typically, what I do is to decide the crystal and loop size even before I open the cover slide. I do it by testing the loop size on top of the unopened cover slip. Then gently open the cover slip and pick up only the crystal I want (hopefully this will be a success). Then quickly close the cover slip on top the original well and close it. If it is done quickly the remaining crystal will stay for another one or two rounds.

Harshith, The VDX plates come in two varieties: 1) plain and 2) pre-greased. The one I show is pre-greased. One can buy Dow Corning high-vacuum silicone grease and manually apply to the lips of the wells. The pre-greased ones are expensive but convenient.

@@thayumanasomasundaram699 I highly appreciate your reply. Thank you sir.

Avvaru, I am not sure what do you mean by three steps of crystallization. In this video I am only showing how to pick up a crystal. There are other videos where I show how to mount the crystal in the cryo stream.

Karima, the holding time varies (10-20 min) depending upon the surrounding temperature. However, you want to keep the dewar filled to top to avoid gradual variation of temperature which will produce ice (bad for your crystals). Let us say room temperature is 300K and LN2 temperature is 77K. You want to go quickly from 300 to 77 rather than through nitrogen gas which may have in between temperature. Note we want quick (flash) cooling and NOT slow cooling. Hope it is clear.

Can you be more specific. What part of the video you don't understand?

Mehvesh,Yes. You can use square cover slips. However, due to the shape the squares may overlap between two wells making it difficult to flip open to retrieve the crystal. Test it out before setting up the trays.

Yifan, Note that I picked and dropped the crystals multiple times so that it is clear in the video for people to see. In real experiment one needs to pick the crystal only once and freeze it. I hope that is clear.

Thayumana Somasundaram OK, i understand. thanks for your nice video.