Q: How do you load a 96 condition screen?

A: The user takes a standard 96-well plate containing the precipitants to be screened and places it on the stage of the Plug Maker. The Plug Maker is then easily programmed to automatically sample the desired precipitants from the 96-well plate and combines them with the protein inside the CrystalCard.

 

Q: Can you design and load your own sparse matrix screen?

A: The Plug Maker will sample any precipitant that the user places in a 96-wll plate on its stage.

 

Q: Can you purchase pre-made screens?

A: Emerald BioSystems will offer all of their standard screens and any custom screen for purchase in the standard 96-well plate format.

 

Q: What does the Plug Maker offer that others don’t?

A: Several answers listed below:

1.       Low volume but useable crystals: The Plug Maker and CrystalCard is a nanovolume crystallization screening system that still produces Diffraction-Ready crystals. The volume of the plugs is small enough (~10nL) to produces thousands of experiments with a few microliters of protein, but large enough to produce crystals of useable size for diffraction experiments (~50-200 um). Further, the crystals can be extracted from the Peel-Apart CrystalCards and translation/scale-up is not required.

2.       Fine gradient screening: The Plug Maker enables efficient and comprehensive optimization of protein/precipitant combinations through fine gradient optimization experiments. Exquisite control of microfluidic flow rates allows the Plug Maker to scan a narrow region of crystallization phase space in order to optimize protein crystals.

3.       Sparse matrix+ gradient screening: The Plug Maker’s version of sparse matrix screening (called the hybrid method) simultaneously produces sparse matrix and optimization screens. This is achieved by generating a concentration gradient over a series of tens of plugs with each precipitant.

4.       Reproducibility: Inasmuch as the crystallization of proteins can be reproducible (considering the fundamentally stochastic nature of protein crystal nucleation), the Plug Maker provides some unique advantages. Subtle factors that can effect crystallization such as mixing style/rate, pipetting error, environmental factors (dust, debris settling on the crystallization plate), and evaporation during setup, etc. are eliminated when using the Plug Maker. Instead, repeated experiments in plugs are mixed in the same way, experience the same environmental factors (being encapsulated in the carrier fluid) and do not experience evaporation during setup because of the enclosed environment.

5.       In situ diffraction: The entire CrystalCard can be mounted on a goniometer for X-ray diffraction analysis of crystals can be done without touching the crystals. Further, it has been shown that data can be collected from multiple crystals at room temperature while they remain in the CrystalCard and combined in order to generate full data sets and solve new protein structures.

 

Q: Are the CrystalCards reusable?

A: The CrystalCards are one-time use consumables, but may be incubated for crystal growth for many months.

 

Q: How should I store the CrystalCard after setup?

A: The CrystalCard should be stored at 100% humidity or the plugs will eventually evaporate. Otherwise, they can be store in any manner that is convenient such as a microscope slide box with a moist paper towel, a plastic box containing a beaker of water.

 

Q: How long can I incubate the CrystalCards?

A: Stored at 100% humidity, the plugs in the CrystalCards should last for at least 6 months (likely much more)

 

Q: Can I use evaporation to my benefit?

A: Of course! By controlling the humidity of the chamber, a controlled evaporation of the plugs may be beneficial in inducing crystal growth.

 

Q: When I get a crystal, how do I know what condition it grew in?

A: It is easy to interpolate the condition that the crystal was grown in as long as the starting and finishing conditions are recorded. Because of the fineness of the gradient, interpolation in the CrystalCard can be extremely accurate.

 

Q: What are common follow-up experiments after an MPCS optimization has been run?

A: Of course, this depends on the initial experiment that was run and its result. 

Result: Lots of large single crystals

Follow-up: Extract and cryo-protect for diffraction experiments or store for future extraction or in situ diffraction

 

Result: One or a few large single crystals

Follow-up: Harvest and cryo-protect crystal(s) for diffraction experiments. run a follow-up optimization with a narrower gradient surrounding the condition that produced the crystal(s), or interpolate the condition and fill a CrystalCard at that condition to produce more crystals.

 

Result: Smaller crystals or microcrystals

Follow-up: Run a follow-up optimization with a narrower gradient surrounding the condition that produced the crystals, interpolate the condition and fill a CrystalCard at that condition to produce more crystals, or run a similar but different gradient varying different components (i.e. the additive, pH, or protein/precipitant ratio).

 

Result: Empty plugs

Follow-up: Increase protein or precipitant concentration or both or run a different gradient that involves the same components such as varying the protein/precipitant ratio.

 

Q: How much do the general MPCS materials cost?

A: The CrystalCard is the primary consumable. Over time, the carrier fluid and the Teflon tubing will need to be refilled and replaced but the cost is negligible.