Hanging drop vapor diffusion method crystallization
Answers
Answer:
Explanation:
The hanging drop vapor diffusion technique is a popular method for the
crystallization of macromolecules. The principle of vapor diffusion is
straightforward. A drop composed of a mixture of sample and reagent is
placed in vapor equilibration with a liquid reservoir of reagent. Typically the
drop contains a lower reagent concentration than the reservoir. To achieve
equilibrium, water vapor leaves the drop and eventually ends up in the reservoir. As water leaves the drop, the sample undergoes an increase in relative
supersaturation. Both the sample and reagent increase in concentration as
water leaves the drop for the reservoir. Equilibration is reached when the
reagent concentration in the drop is approximately the same as that in the
reservoir.
Benefits of Hanging Drop Crystallization
• Can be cost effective.
• Sample and reagents in contact with a siliconized glass surface.
• Easy access to crystals.
• Can perform multiple drops (experiments) with a single reservoir.
Using the VDX Plate for Hanging Drop Vapor Diffusion
The VDX Plate is a 24 well plate manufactured from clear polystyrene. The
VDX Plate is typically sealed with High Vacuum Grease and Siliconized 22
mm Circle or Square Glass Cover Slides. Rows of the plate are labeled A-D
and columns are labeled 1-6 on the VDX Plate.
Applying Sealant to a VDX Plate
1. Apply a bead of High Vacuum Grease along the top edge of the raised reservoir A1 of the VDX Plate. It is recommended that one apply the high vacuum
grease prior to pipetting the reagent. Create a circular bead on the upper
edge of the reservoir. Do not complete the circle. Leave a 2 mm opening
between the start and finish of the circular bead. Leave a 2 mm opening between the start and finish of the circular bead as this will allow air pressure
to escape when sealing the reservoir with a cover slide. For convenience, time
savings and a consistent seal, the VDX Plate is also available with sealant.
2. Pipet 1.0 milliliter of crystallization reagent into reservoir A1 of the VDX
Plate. (Note: Recommended reservoir volume is 0.5 to 1.0 milliliters)
3. Clean a Siliconized 22 mm Circle or Square Cover Slide by wiping the
cover slide with lens paper and blowing the cover slide with clean, dry compressed air. Pipet 2 microliter of sample into the center of a Siliconized 22
mm Circle or Square Cover Slide. (Note: Recommended total drop volume
is 2 to 20 microliters)
4. Pipet 2 microliter of reagent from reservoir A1 into the drop on the cover
slide containing the sample. (Note: Some prefer to mix the drop while others
do not. Proponents of mixing leave the pipet tip in the drop while gently aspirating and dispensing the drop with the pipet. Mixing ensures a homogeneous drop and consistency drop to drop. Proponents of not mixing the drop
simply pipet the reagent into the sample with no further mixing. Not mixing
allows for the sample and reagent to mix by liquid diffusion).
5. Holding the cover slide with forceps, the Pen-Vac, or on the edge between
your thumb and forefinger, carefully yet without delay invert the cover slide
so the drop is hanging from the cover slide.
6. Position the cover slide onto the bead of grease on reservoir A1. Gently
press the slide down onto the grease and twist the slide 45º to ensure a complete seal.
7. Repeat for reservoirs 2 (A2) through 24 (D6).
Hanging Drop Tips
• Note the VDX Plate has a raised cover to protect the cover slides during
transport and storage.
• To access a drop and/or reservoir simply grasp the edge of the cover slide
with forceps or fingertips, twist and pull gently while holding the plate
steady. The Thick (0.96 mm) cover slides resist heavy handling better than
the regular (0.22) cover slides.
• VDX Plates can be stacked for convenient storage.
• One can pipet multiple drops onto the cover slide. This technique is often
useful when screening additives since one can use the same reservoir with
multiple drops with each drop containing a different additive. This technique can also be used to screen different drop sizes and ratios versus the
same reservoir. Use care not to avoid mixing the drops during pipetting,
plate transport, and plate viewing.
• Label plates with protein name, experiment, drop & reservoir volumes,
and date.
• Use the Glass Cover Slide Gizmo Dispenser for fast and easy handling of
siliconized cover slides. Or try the Cover Slide Vacuum Gadget (HR8-098) or
Pen-Vac (HR3-251) for holding and manipulating cover slides.
Solutions for Crystal Growth
Reservoir Solution
H2O
[ppt]
drop =
[ppt]
reservoir
2
Figure 1
Process of vap