Instructions for manual use of Vapor Batch crystallization plate

Suitable for microbatch and vapor diffusion crystallization.

Thank you for using the Vapor Batch Crystallization Plate.  This plate can be used in several different ways:

A. Screening with microbatch

1. Flood the sample area of the plate with 2.5 ml of a 50:50 mixture of silicone oil (Dow Corning 200/1 cs) and paraffin oil (e.g. Merck’s “Paraffin Highly Liquid”)

2. Dispense around 1 of screening cocktail through the oil into each sample well.

3. Dispense around 1µl of protein into each sample well, ensuring that it mixes with the screening solution.  

4. Replace the lid on the plate.

5. Incubate.

Trials have shown that microbatch is more efficient than vapor diffusion at picking up crystallization hits (e.g. Baldock et al., 1996; D’Arcy et al.,2000; see also results compiled by Douglas Instruments). The hydrophilic version of the plate VBATCH 1/1 PHI-80 is recommended for screening (especially with drops below 0.5 µl) because the drops adhere to the plate better.

B. Optimization with microbatch

Follow the same procedure as for screening, but use pure paraffin in step 1.  Pure paraffin minimizes evaporation, which makes it easier to interpret experiments.  Around 50% of proteins give crystals that diffract better when grown using microbatch than using vapor diffusion.  The hydrophobic version of the plate VBATCH 1/1 PHO-80 is recommended for optimization experiments because it reduces crystal nucleation and crystal sticking to the plastic.

C. Screening and optimization with vapor diffusion

1. Flood the sample area of the plate with 2.5 ml of pure silicone oil.

2. Dispense around 1µl of screening cocktail through the oil into each sample well.

3. Dispense around 1µl of protein into each sample well, ensuring that it mixes with the screening solution.

4. Dispense around 8 ml of e.g. 0.5 M ammonium sulfate solution into the reservoir wells around the outside of the plate.  (Distribute the solution fairly evenly around the wells.)

5. Remove 1.5 ml of the silicone oil and discard,

6. Place a bead of silicone or paraffin grease around the inside of the rim of the lid. Replace the lid on the plate, ensuring that a good seal is created.  Bending up a corner of the lid as you lower it can help.  (Or seal with tape).

7. Incubate.

If no crystals grow, or if precipitation is in general very light, try increasing the concentration of the reservoir solution in steps.

Notes.

1. The silicone oil used in microbatch screening allows slow evaporation.  This gives a result which is similar to vapor diffusion in that it scans through many crystallization conditions.  The proportion of silicone oil can be adjusted – using more silicone increases evaporation, using less reduces it.

2. If you use less than 1µl +1µl drops in microbatch experiments, reduce the proportion of silicone oil.

3. If you dispense automatically, accuracy can be improved by dispensing the protein and cocktail solution first, then covering with 10 µl of paraffin oil straight away.  This method can be used with Oryx4 or 8 but not with IMPAX.

4. If you get crystals of salt, try increasing the volume of protein or decreasing the volume of screening cocktail.

5. For maximum crystal nucleation, do not stir the droplet.  To reduce nucleation, stir.

6. The silicone/paraffin mixture (and VB plates) can be obtained from Hampton Research under the trade name “Al’s Oil”.

7. To store microbatch crystals, dispense salt solution or water in the reservoir wells and seal with grease.

8. Holders are available to convert the footprint of the plate to a regular Linbro or SBS (96-well) footprint.  

9. Douglas Instruments is running a competition for the best use of the Douglas Vapor Batch Plate.