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Crystal Former - Original and Scale-up Formats
What is the minimum volume of sample per channel?
The total volume of the channel is 150nL. While some liquid handling robots are capable of dispensing such small volumes, drop sizes of at least 0.3 µL each for the protein and precipitant are recommended. For total drop volumes of 0.5 µL, the sample consumption for the Crystal Formers is comparable to most automated screening methods.
What protein concentration should I start with for my initial screening?
We recommend starting with the protein concentrations between 60-80% of saturation point. The saturation point can be estimated as the point where your protein begins to precipitate during concentration process. This might correspond to 10-30 mg/ml for reasonably well-behaved proteins, but could be significantly lower for poorly-soluble ones. If you are trying to reproduce the vapor diffusion hit, you we recommend increasing your protein concentration.
What are the recommended sample volumes?
For the Original Crystal Former, it is recommended that the protein volume be between 0.3-0.5 µL. The corresponding volume for the crystallization solution typically ranges between 0.3 – 1 µL. The microchannels of the Scale-up chip are larger, thus 1.5 µL of protein sample with 0.5-1.5 µL of crystallization solution should be loaded per channel.
How do I know if the channels have filled with protein?
If using colorless protein sample, channels are best visualized on a black background; for colored proteins use white background. If plates are set up manually, channel filling can be confirmed by monitoring the solution movement across the channel immediately after drop application. For most protein samples this takes 1-2 seconds. For samples containing glycerol or other viscous additives, the process might take a bit longer. Please refer to our video demonstration of a manual chip set up
How do the microchannels vary between the Crystal Former formats?
For all chips, the length of the microchannels is 10 mm. Both the 16- and 96-channel original Crystal Formers have channels that are 150 nL in total volume. In contrast, the microchannels of the Scale-up Crystal Former are 1.1 µL in total volume.
Can I use an alternative sealing method?
Sealing tape is provided in a dual-strip format for sealing only the inlet wells. This design minimizes potential imaging difficulties that arise from air bubbles trapped between the tape and the microchannels when larger tape strips are used. When sealing the Crystal Formers, the user should avoid pressing with excessive force, as this will cause mixing of the reactions and dissipate the gradient. Microlytic’s format for sealing tape thus also avoids placing undue pressure over the microchannels. Any other sealing film regularly utilized for sealing crystallization trials is, in principle, compatible with the Crystal Formers, though it is recommended that the substitute tape be trimmed such that it only covers the sample inlets.
Can the Crystal Formers be used to crystallize my integral membrane protein?
Yes. The materials used to manufacture the Crystal Formers are fully compatible with high concentrations of detergents used in the solubilization of membrane proteins. Keep checking back for the final published reference highlighting the successful crystallization of an integral membrane protein with the Crystal Former.
Can I use organic solvents in the Crystal Former?
Yes. To date, we have not identified any common organic compounds used in protein crystallization that are incompatible with the Crystal Formers.
How should I store the Crystal Formers?
Crystal Formers do not require any special storage conditions. For extended incubation periods, higher humidity storage (>60%) is recommended to minimize evaporation. This can be easily achieved by storing the Crystal Formers in a small container that also includes a moist napkin.
Can the Crystal Formers be used for in situ testing?
All formats of the Crystal Former are compatible with in situ diffraction testing, providing that a suitable chip mount is available. The Crystal Formers are also compatible with the PX Scanner from Oxford Diffraction (Read the Whitepaper).
Are the Crystal Formers compatible with my UV imaging system?
The materials used in Crystal Former manufacturing are fully compatible with UV imaging. Please refer to our whitepaper for additional information.
Are the Crystal Formers automatable?
The 16-channel formats are not recommended for automated use. We refer the user to our new SBS High Throughput Crystal Former which has been specifically designed for robotic compatibility and lower protein consumption.
How long does equilibration of the channel take?
Equilibration of ions in the microchannel takes approximately 1 week. Larger polymers and macromolecules will diffuse more slowly and may thus take up to two weeks for full equilibration. Equilibration is not necessary, however, for crystal growth and many crystals appear rapidly (18-72 hours) within the microchannels.
Will I get crystals faster using Crystal Former compared to vapor diffusion?
Liquid-liquid diffusion provides higher crystallization success rate due to more thorough sampling of the protein phase diagram that the vapor diffusion. However, ultimate success is dependent on your target. For challenging targets that have been screened extensively in vapor diffusion and resisted crystallization for months or years, it will likely be necessary to perform more than one screen in Crystal Former. Same vapor diffusion screens may be utilized, as different mixing kinetics and the power of the concentration gradient may allow for a discovery of new crystallization condition(s) and/or new crystal morphologies. For new targets, we recommend setting up at least a 48-condition SuperSMART and a 48-condition PurePEG screen in parallel to your regular vapor diffusion screens. You may also use your own vapor diffusion screens in the Crystal Former. For targets that yielded poorly diffracting crystals in the vapor diffusion, we recommend re-screening at least our 48-condition SuperSMART set in the Crystal Former in addition to a standard grid-screen optimization.
I did not get any crystals after my initial screen, what do I do?
Estimate the percentage of the channels that contained any precipitate. If the majority of your channels are completely clear, increase the protein concentration and repeat the screen. If a substantial number of channels contain some precipitate, inspect those channels for precipitation pattern. Heavier protein precipitation is expected next to the precipitant inlet, and lighter-to-no precipitation is expected towards the protein inlet. Absence of any precipitation patterns suggests that the gradient was dissipated during the set-up procedure. This is most often caused by applying too much pressure to the sealing tape. If using your fingertip to seal the inlets, your fingernail should NOT turn lighter from the pressure. Please refer to our video demonstration of a manual chip set up
How do I harvest crystals from the microchannels?
Crystals are harvested from the back of the Crystal Former. Use a sharp blade to cut the sealing film on either side of the microchannel and peel the film away. To facilitate crystal manipulation, use a loop that is no larger that 100 µm, as this will permit complete submersion of the loop into the channel. The addition of a stabilization solution on exposure of the channel to air is recommended. The stabilization solution can be estimated based on the history of crystal appearance along the channel and the incubation time. As equilibration of the channel typically takes 1 week (for a salt), samples that are at least 1 week old can be stabilized at approximately 50% of the starting crystallization solution.
Are there any recommended protocols for crystal cryoprotection?
The harvesting and cryoprotection of a protein crystal grown in any given system frequently requires some degree of carefully consideration and optimization. Once crystals have been harvested from the microchannels of the Crystal Former, they may be manipulated using standard methods. As mentioned above, crystals may be stabilized using a solution that approximates the equilibrated condition. Alternatively, the cryoprotectant or cryoprotectant-mother liquor combination may be applied directly over the channel and the crystals flash frozen immediately on extraction. When the conditions for cryoprotection are unclear, mineral oil or paratone oil may be used to cover the exposed microchannel and the crystals again flash frozen after extraction through the oil layer.
The design of the Crystal Former also presents a unique opportunity for the gentle cryoprotection and/or dehydration of crystals. The cryoprotectant can be applied to one of the sample inlets and allowed to diffuse through the microchannel, thus permitting slow diffusion of the cryoprotectant into the crystal channels prior to harvesting.
How do I optimize a crystallization hit? Can crystallization conditions be translated to other crystallization systems?
Many of the crystallization hits can be translated into vapor diffusion systems by systematic grid screening using the initial crystallization condition as a starting point. We also offer the Crystal Former XL, in which the larger channels place less restriction on the maximum growth of the crystals. Many users have successfully optimized their crystals for data collection by altering the ratio of protein to crystallization solution in the Crystal Former. The resultant change in the crystallization gradient has direct impact on the nucleation rate and thus significantly
I’d like to optimize a condition from a PurePeg crystallization condition, how do I go about this?
You can purchase the individual, USP grade PEG components directly from Microlytic. The PEG cocktail is also available for purchase. Optimization of crystallization hits from the PurePeg screen, however, is not necessarily contingent on use of the PEG cocktail. For many PEG-containing conditions, the identity of the PEG is not absolute for crystal reproducibility. PEG compounds can often be substituted for one another, though a wider range of PEG concentrations should be explored. The major consideration for the reproducibility of crystal hit from the PurePeg screen is the PEG purity. Most PEG compounds utilized for crystallization have not traditionally been USP grade. As such, the presence of contaminants in low PEG grades may hinder the reproducibility of crystals.
Are individual crystallization conditions available for purchase?
Yes, you may order any of the crystallization conditions from the SmartScreen and PurePeg screen.
Why are there only 96 available crystallization conditions? I usually start with many more than that for my initial crystallization trials?
The Crystal Former is designed to enable gently diffusive mixing of protein and precipitant inside microchannels resulting in improved mixing kinetics and significantly higher crystallization hit rates. Liquid-liquid diffusion, harnessed in the Crystal Former, is an orthogonal approach to other methods (e.g. Vapor diffusion, microbatch and dialysis) and samples a significantly different region of the protein phase diagram. When coupled with crystallization conditions that have been optimized for liquid-liquid diffusion approaches, the Crystal Former system provides a robust and efficient method for crystallization screening. The user therefore requires fewer crystallization conditions with significantly increased probability of crystallization success!
How do the SH-1 and SH-2 Crystal Former holders differ from one another?
The SH-1 holder offers a snap-fit mechanism that holds the individual 16-channel Crystal Formers tightly in place. It is fully compatible with many automated systems. The SH-2 holder is a low profile design that is compatible with the visible light objective of the Rock Imager from Formulatrix and is also compatible with all automated systems previously using the SH-1 holder. The SH-1 holder is NOT compatible with the Rock Imager. The SH-2 holder design also affords 2 major advantages: (1) manual manipulation of the Crystal Formers is facilitated by the drop-in design and (2) the reduction in mechanical stress decreases the likelihood of damage to the Crystal Formers during staging. We strongly recommend that users wishing to automate their use of the Crystal Former use only the new SBS High Throughput Crystal Former (CF2-HT).
What is the XZ plate?
The XZ plate has been designed to permit high throughput dialysis experiments. Each well has a small dialysis chamber at the base of the plate that is separated from the main compartment by a dialysis membrane of specified molecular weight cut-off. Sample is delivered into the dialysis chamber by a vacuum-driven microfluidic circuit. The dialysis buffer is introduced by pipetting the buffer into the main chamber.
What additional equipment do I require to use the XZ system?
The system requires a vacuum pump with an ultimate vacuum less than 2mmHg, which is equal to 2 torr, 2.7 mbar, or 29.9 inHg. The vacuum plate pump connector, disposable needles and purge liquid are provided with the XZ Dialysis kit. Many in-house vacuum pumps are compatible with this system. A compatible pump is also available through Microlytic (Cat. # XZ-VP).
Can I use the vacuum of lower strength than 2 torr?
Yes, you can use the vacuum anywhere in the 2-8 torr. Using weaker vacuum will result in slower protein flow through the microfluidic network, so time guidelines provided in the user manual should be extended. The complete plate filling can be confirmed by removing the vacuum adapter from the plate vacuum inlet. Presence of liquid at the bottom of the inlet indicates that the protein has filled the entire plate. If using older-model adapter that makes contact with the bottom of the vacuum inlet, you will observe the extra protein coming out of the plate into the vacuum tubing. It is important to wash or replace the tubing to prevent clogging with dried-up protein.
What components are provided in the XZ Dialysis Kit?
The kit contains 9 XZ plates (3 screening plates, 3 optimization plates, 3 growth and soaking plates), the vacuum tip with tubing adapter, 10 disposable needles, purge liquid, and the recommended XZ crystallization screen.
How do the plate formats differ from one another?
The available plate formats differ in the volumes of sample and crystallization solution. With regard to sample consumption, the screening plates (XZ-S-96) require 6 uL of total protein sample to screen against 96 conditions. This equates to 62.5 nL of protein sample per experiment. The optimization plates (XZ-O-96) require 15 µL of protein sample per 96 conditions, which is equivalent to 156 nL per experiment. Growth and optimization plates (XZ-G-96) use 36 uL of protein per plate, or 375 nL of sample per experiment. Finally, the largest-scale plate (XZ-G-24) requires 25 uL for 24 crystallization conditions, or approximately 1 µL per experiment.
Both the screening and optimization plates use 50 µL of crystallization solution per well. The 96-well and 24-well growth plates require 160 µL and 500 µL, respectively.
While all plates permit crystal harvesting, the larger formats provide more working area and thus easier harvesting. The increased volumes also permit growth of significantly larger crystals for diffraction studies.
How should I prepare the protein sample for XZ screening?
Centrifuge your protein sample to remove any precipitate immediately prior to plate set-up to insure that protein precipitate will not block the micro fluidic network. Blockage of the channels will impede the protein sample flow and result in air bubble formation in the wells. Poorly-soluble ligands added directly to your protein sample will have the same effect.
How is the XZ plate set-up?
The crystallization solution is first added to each of the wells. The plate is then placed under vacuum. The protein solution is then pipette onto the sample inlet film and the film punctured with one of the included needles. At this point, the microfluidic channel will fill evenly with the applied protein sample. Finally, purge solution is applied to the sample inlet and permitted to traverse the microfluidic circuit. The wells are then sealed and the experiment incubated at the desired temperature.
How are the reaction chambers sealed from neighboring ones?
The purge solution displaces the protein sample from the main microfluidic circuit, into the dialysis chambers and seals the chambers to prevent cross contamination during the experiment.
Why are there so many dots at the bottom of the well?
Each well contains a single dialysis chamber. The additional translucent dots comprise a fiducial ring that facilitates inspection of the experiments. A translucent indicator of the row and column number is also present as part of the fiducial ring to facilitate inspection.
Why are the plates black?
Imaging a small dialysis experiment under 50-500 µL of crystallization solution using completely translucent/transparent plates can make inspection of the reaction very difficult. The non-transparent black plate greatly improves imaging of the experiments.
I observe bubbles coming out of the protein sample inlet upon membrane puncturing - what went wrong?
Air bubbles coming out of the protein inlet upon membrane puncturing indicate that the plate was not under vacuum at that time. This usually occurs when there is an air leak in the vacuum tubing, or when the fine tubing connecting the plate to the main vacuum line gets clogged with dried-up protein sample. We recommend washing out the tuning after each set of experiments, or using fresh tubing to prevent this from happening.
I am having difficulty imaging the plates due to condensation on the sealing film – what should I do?
Some crystallization conditions result in the accumulation of condensation on sealing tape. This is exacerbated when plates are stored in areas where the temperature and humidity fluctuate. The simple solution is to quickly replace the sealing film with a spare film immediately prior to imaging. As the method relies on the diffusion of liquids across the dialysis membrane, the crystallization experiment is not severely impacted.
How was the Salt-in, Salt-out™ screen developed?
The crystallization conditions of the Salt-in, Salt-out™ screen have been selected for optimal performance with the dialysis method. Dialysis is the only crystallization technique that allows crystallization both in the salting in (low ionic strength) and salting out (high ionic strength) regions of the phase diagram. This screen thoroughly explores the protein phase diagram in both the salting in and salting out regions. The screen is based on analysis of crystallization conditions that have been successful in dialysis-based protein crystallization for more than 100 different proteins, including integral membrane proteins, protein complexes, viruses, and soluble proteins.
Can I use my own crystallization screens with the XZ plates?
If using other crystallization screens, it is important to note that precipitants often using in other crystallization formats will not diffuse readily across the dialysis membrane. Polyethylene glycol of molecular weight 8000 or higher will be excluded from the crystallization chamber. PEGs with concentrations greater that 10% with also exhibit sub-optimal equilibration across the membrane and should be used with caution.
What are the applications of the XZ plate?
The XZ system has been designed for dialysis-based protein crystallization. However, as the system effectively permits efficient, high throughput dialysis, it can be used for many biochemical applications, including buffer screening for protein solubility and stability.
I think I have some crystals – what now? Can I harvest from these plates?
The XZ plates can be directly mounted in a plate holder at any beamline that is capable of this sort of in situ screening. Alternatively, crystals can be extracted directly from the dialysis chamber. Using a pair of tweezers, the dialysis membrane can be removed from the top of the dialysis chamber while the precipitant solution is still in place. You may wish to remove some of the crystallization solution prior to opening the dialysis membrane to prevent the crystal from floating into areas of the well that are difficult to image.
Are the XZ plates compatible with automation?
Yes, the XZ plates are SBS format and can therefore be programmed for use with a variety of liquid handling and imaging robots. The plates can also be used for in situ diffraction screening on beamlines equipped with appropriate SBS-format plate holders. Please contact our technical support line for further information.