Hanging Drop Vapor Diffusion Protein Crystallization Tutorial

In this tutorial, I will demonstrate how to set-up a hanging drop vapor diffusion protein crystallization set-up. Hanging drop crystal set-up is a common procedure for growing protein (macromolecular) crystals. In this demonstration I will show all the components needed: VDX Pre-greased plate, siliconized cover slip, pipettes, tips, protein, precipitant and other materials. Then in the second section I will show how to set-up 3 micro liters of protein (here enzyme lysozyme) and mix it 3 micro liters of a precipitant (here 10% sodium chloride) on a siliconized cover slip and seal it over a precipitant solution.

[@user-tp2vc1ut4s]

this video is super helpful! Thank you!

[@nathaliasilva5368]

amazing

[@bhagyasriboddu6665]

Here what is the precipitated solution?

[@thayumanasomasundaram699]

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.

[@mr.harambae]

what is it greased with?

[@thayumanasomasundaram699]

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.

[@mr.harambae]

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