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发表于 2009-9-6 14:43:31
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The sitting drop vapor diffusion technique is the 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 Sitting Drop Crystallization
• Allows one to avoid greasing plates.
• Can be cost effective.
• Can be time efficient.
• Often easier when using detergents and hydrophobic reagents.
• Drops can be positioned in a stable sitting position.
• Compatible with gels.
Using the Cryschem Plate
The Cryschem Plateª is a 24 well plate manufactured from clear polystyrene. Each
well contains a post in the center which is elevated above the bottom of the reservoir.
The smooth, concave depression in the post can hold up to 40 microliter drops
and the reservoir can hold up to 1.2 microliters of reagent. The Cryschem Plate is
sealed with either Clear Sealing Tape or Plain 22 mm Circle or Square Glass Cover
Slides. Rows are labeled A-D and columns are labeled 1-6 on the Cryschem Plate.
1. Pipet 0.5 milliliter of crystallization reagent into reservoir A1 of the Cryschem plate.
(Note: Recommended reservoir volume is 0.5 to 1.0 milliliters)
2. Pipet 1 microliter of sample into the post of reservoir A1. (Note: Recommended
total drop volume is 1 to 40 microliters)
3. Pipet 1 microliter of reagent from reservoir A1 into the drop in post A1. (Note: Some
people 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 homogenous drop and consistency drop to drop.
Proponents of not mixing the drop simply pipet the reagent into the sample with
no further mixing).
4. Repeat steps 1 through 3 for the remaining 23 reservoirs.
5. Seal the Cryschem Plate with 2 strips of Clear Sealing Tape (HR4-510).
Cryschem Plate Tips
• Use Crystal Clear Sealing Tape. Other Brands are optically inferior and can turn
opaque in the presence of certain crystallization reagents.
• To access a drop and/or reservoir of a Cryschem Plate sealed with tape simply
make a circular incision in the tape using the inside of the reservoir as a guide. Use
a sharp blade to cut the tape and hold the incised piece of tape with forceps. The
opening can be sealed with another strip of tape or a plain 22 mm circle or square
cover glass and high vacuum grease.
Using Micro-Bridges
The Micro-Bridge¨ is a small bridge (inverted U) manufactured from clear polystyrene
and clarified polypropylene which contains a smooth, concave depression in
the center of the top region of the bridge (figure 3). The Micro-Bridge can hold up
to 40 microliter drops. The Micro-Bridge is
inserted into the reservoirs of VDX, Linbro, or
Costar Plates to perform a sitting drop vapor
diffusion experiment. The design of the Micro-
Bridge is such that the bridge is quite stable in
the reservoir and does not require the Micro-
Bridge to be bonded to the plate. The Micro-
Bridge can be removed from the plate for crystal
manipulation and observation if desired.
1. Pipet 1.0 milliliter of crystallization reagent into reservoir A1 of a VDX, Linbro, or
Costar plate. (Note: Recommended reservoir volume is 0.5 to 1.0 milliliters)
2. Place a clean (blow the Micro-Bridge with clean, dry compressed air before use)
Micro-Bridge into the bottom of reservoir A1 such that the concave depression in the
Micro-Bridge is facing up.
3. Pipet 2 microliters of sample into the Micro-Bridge in reservoir A1. (Note:
Recommended total drop volume is 1 to 40 microliters)
4. Pipet 2 microliters of reagent from reservoir A1 into the drop in the Micro-Bridge
A1. (Note: Some people 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 homogenous drop and consistency drop to
drop. Proponents of not mixing the drop simply pipet the reagent into the sample
with no further mixing).
5. Repeat steps 1 through 3 for the remaining 23 reservoirs.
6. Seal the plate with 2 strips of Clear Sealing Tape (HR4-510). The VDX, Linbro, and
Costar Plates can also be sealed using sealant and plain glass cover slides.
Micro-Bridge Tips
• Use Crystal Clear Sealing Tape. Other Brands are optically inferior and can turn
opaque in the presence of certain crystallization reagents.
• To access a drop and/or reservoir of a sealed with tape simply make a circular
incision in the tape using the inside of the reservoir as a guide. Use a sharp blade to
cut the tape and hold the incised piece of tape with forceps. The opening can be
sealed with another strip of tape or a plain 22 mm circle or square cover glass and
figure 2 crystallization droplet
(2 μl Sample / 2 μl Reagent)
H2O H2O
[ppt]drop =
[ppt]reservior
2
figure 1
Process of vapor diffusion.
figure 3
Sitting Drop Vapor Diffusion
Crystallization
Crystal Growth 101
Solutions for Crystal Growth
Sitting Drop Vapor Diffusion
Crystal Growth 101
Solutions for Crystal Growth
Principle
A drop composed of a mixture of sample and reagent is placed in vapor equilibration
with a liquid reservoir of reagent. Over time, a reagent concentration equilibrium is
reached between the drop and the reservoir (figure 1).
If the sample is to be crystallized using
salts, polymers, non-volatile organics, and
pH, the drop will typically contain 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 in the drop
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.
If the sample is to be crystallized using volatile organics, the drop can contain either
a lower or higher initial reagent (volatile organic) concentration than the reservoir.
In other words, one can choose to vapor diffuse volatile organics from the reservoir
to the drop by having a higher concentration of volatile organic in the reservoir
than the drop. Or one could have a lower concentration of volatile organic in the
reservoir compared to the drop and the volatile organic would diffuse from the drop
to the reservoir.
If the sample is more soluble in a higher concentration of volatile organic (typical),
then one would begin the vapor diffusion experiment with the drop containing a
higher concentration of volatile organic than the reservoir. As the volatile organic
leaves the drop for the reservoir, the relative supersaturation of the sample in the
drop would increase.
If the sample were less soluble in a higher concentration of volatile organic (atypical),
then one would begin the vapor diffusion experiment with the drop containing
a lower concentration of volatile organic than the reservoir. As the volatile organic
leaves the reservoir for the drop, the relative supersaturation of the sample in the
drop would increase.
Other scenarios are possible, especially if one screens reagent systems which are
mixed with volatile organics and salts and/or polymers. Here, one must consider
both the equilibration path of the volatile organic and the non-volatile salt and/or
polymer.
Clarified Polypropylene VDX Plate for Sitting Drop Vapor Diffusion Crystallization
1. Using a single Clarified Polypropylene VDX Plate, place a single Clarified
Polypropylene Micro-Bridge in each reservoir. See figure 2 below.
2. Apply a bead of high vacuum grease to the rim of each of the 24 reservoirs.
Apply the bead in a smooth circular motion, leaving a small gap, creating an incomplete
circle in the bead of grease. This gap will allow trapped air to escape from the
reservoir when the cover slide is applied. When sealing the cover slide to the reservoir,
simply place the cover slide on the bead of grease, press gently, then twist
approximately 30 degrees or more to seal the gap. See figure 3.
3. Pipet 1.0 milliliter of the crystallization reagent into reservoir A1 of the
Polypropylene VDX Plate.
4. Pipet the sample, solubilized in the volatile organic, into the depression of the
Polypropylene Micro-Bridge. The Polypropylene Micro-Bridge can hold up to 40
microliters of liquid.
5. Seal the reservoir with a 22 mm diameter plain glass cover slide (22 mm circles or
squares may be used to seal the reservoir).
6. Repeat steps 1 through 5 for the remaining 23 reservoirs. In the remaining reservoir
one may evaluate the effects of various additives, varying concentrations of the
volatile organic, pH, or other chemical crystallization variables.
Temperature as a Crystallization Variable
If it is suspected that the sample might have temperature dependent solubility, the
crystallization screen or reagent conditions may be repeated at one or more additional
temperatures below or above room temperature.
Making Changes to the Reservoir and Drop
Additions, deletions, or modifications to the drop and reservoir can be performed by
simply removing the glass cover slip, performing the modification and then resealing
the cover slide to the reservoir.
Compatibility with Volatile and Aggressive Organic Solvents
The clarified polypropylene used in the Polypropylene VDX and Polypropylene
Micro-Bridges are compatible with a wide number of volatile and aggressive organic
solvents. However, to be certain of compatibility, it is recommended that the organic
solvent (or reagent) to be used for crystallization be tested with the Clarified
Polypropylene VDX Plate and Clarified Polypropylene Micro-Bridge prior to setting
the experiment to confirm compatibility. Pipet 1 milliliter of the reagent into the
reservoir. Seal with a glass slide and vacuum grease. Observe for compatibility.
Vapor Diffusing Volatile Organics Out of the Drop Into the Reservoir
If the sample is more soluble in high rather than low concentrations of volatile
organic, then one might consider vapor diffusing the volatile organic out of the crystallization
drop. In such an experiment, the reservoir should initially contain a con-
For Small Molecules & Peptides
[ppt]drop =
[ppt]reservior
2
H2O H2O
figure 1
figure 2
well of a Clarified
Polypropylene VDX
Clarified Polypropylene crystallization plate
Micro-Bridge
reservoir solution
figure 3
22mm plain circle or
square cover slide
reservoir solution
vacuum grease |
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