The Crystal Former XL (Scale-up) is ideal for the reproduction and optimization of crystallization conditions obtained in the High Throughput or Original Crystal Formers as the crystallization microchannels of the Scale-up chip are larger than those of the screening formats. 

 

As for other Crystal Former formats, the Scale-Up version is a microfluidic device for protein crystallization that exploits the favorable physical conditions occurring at the microscale.  This environment, when combined with the Crystal Former's special materials, unique surface treatment, and proprietary mixing kinetics substantially improve crystallization success.

 


Key advantages:

  • Liquid-liquid diffusion to generate crystallization gradients
  • Simple to use, no external equipment is required, ready to add to any workflow
  • Ideal for crystal optimization
  • Direct Access to crystals for harvesting

 

This format comprises 16 microchannels on the footprint of a standard microscope slide. Each microchannel is  270 microns x 400 microns x 10 mm with a channel volume of 1.1 µL. The Crystal Former Scale-up is compatible with the SBS-format adapters SH-1 and SH-2 for easy storage.

 

The Crystal Former Scale-Up can be operated with standard pipettes.  After loading precipitant and protein solutions, the microfluidic architecture encourages proper mixing and separation of individual crystallization reactions.

 
To use the Crystal Former, first add protein into an inlet of choice; capillary effects will cause the channel to fill. Once protein loading is complete, crystallization reagents are added to the opposing inlet to form a liquid-liquid interface. As the two solutions equilibrate, the microchannel design minimizes the natural convection of the system to allow optimal diffusive mixing of protein and crystallization reagent. 

 

 

Are the crystals useable?

The Crystal Former allows users to harvest crystals grown inside the chip through a removable rear foil..  This enables collection of diffraction patterns from crystals grown in the Crystal Former without the need to repeat experiments in larger volumes, or using different techniques.  Direct access also permits smaller crystals to be harvested for subsequent seeding experiments.

 
Individual Crystal Formers
SKU
Crystal Former Scale-Up (20 pk)

The Crystal Former XL (Scale-up) is ... Read more

CF-XL-20 $600.00
Original Crystal Former (20 pk)

The Original Crystal Former is a microfluidic device for protein crystallization that exploits... Read more

CF-O-20 $600.00
SBS High Throughput Crystal Former - 10 pack

The Crystal Former is a microfluidic device for protein crystallization that exploits the... Read more

CF-HT2-10 $1,150.00
SBS High Throughput Crystal Former - case of 50

The Crystal Former is a microfluidic device for protein crystallization that exploits the... Read more

CF-HT2-50 $5,000.00
High Throughput Crystallization Kit for Mosquito & NT8

A comprehensive starter kit for diffusive mixing compatible with both... More

MCK2-HT-M $1,250.00
High Throughput Crystallization Kit

A comprehensive starter kit for diffusive mixing compatible with both... More

MCK2-HT $1,250.00
MiTeGen Crystal Harvesting Kit - Small This starter kit for small crystal harvesting has been assembled by the MiTeGen... More MiTeGen-Small $156.50
MiTeGen Crystal Harvesting Kit - Large This starter kit for small crystal harvesting has been assembled by the MiTeGen... More MiTeGen-Large $339.50
Drop-In Holder Adapter for the 16-channel Original and Scale-Up Crystal Formers SH-2 $100.00
Sealing Tape for Crystal Former Original and Scale-Up

A roll of 100 pre-cut, clear sealing strips designed to ensure inlet sealing... More

RT-2 $25.00
Magnetic Crystal Former Mount for in situ screening CF-MM $25.00

How do the microchannels vary between the Crystal Former formats?

The 96-channel Crystal Former channels are U-shaped, are 10 mm in length, 100 x 150 µm in diameter, and 150 nL in volume. The original 16-channel Crystal Former channels are linear, 10 mm in length, and 100 x 150 µm in diameter, and 150 nL in volume. The 16-channel Scale-up Crystal Former channels are linear, 10 mm in length, 270 x 400 µm in diameter, and 1.1 µL in volume.

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 0.3 µL each for the protein and precipitant are recommended. For manual pipetting, minimum volumes of 0.5 µL are recommended for beginners, and 0.3 µL for experienced users.

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 of average size, but could be significantly lower for poorly-soluble ones. If you are trying to reproduce or optimize a vapor diffusion hit, we recommend increasing your protein concentration.

How do I know if the channels have filled with protein? I

f 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 visualizing 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 does Crystal Former sealing method differ from regular sitting-drop vapor diffusion plates?

Whole-plate sealing film is provided with Crystal Former plates. It is made with optimized contact adhesive that does not require much pressure to seal the plate. User should avoid pressing the film with excessive force, as this will cause mixing of the reactions and dissipate the gradient. Any other sealing film regularly utilized for sealing crystallization trials is compatible with the Crystal Formers. A silicone plate sealing roller may be used to apply even pressure to the sealing film. Regardless of the seal type used, the most important aspect of Crystal Former sealing is to avoid bubble trapping under the film, as it will cause solution movement within the channels and gradient mixing. To accomplish this, gently place one edge of the sealing film over the edge of the plate, then use one hand to gently lay the rest of it, while simultaneously using the other hand to smooth the tape over the plate. One should not run one’s fingers/thumb over the plate to ensure sealing: this is not only unnecessary but can increase the possibility of solution mixing/gradient dissipation.

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.

Can I use oils in the Crystal Former?

Most crystallization oils are compatible with the Crystal Former and can be used safely in crystal formers for at least one week. Notable exceptions are Al’s Oil, Paraffin Oil and Silicon oil: these should not be used with the crystal former.

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 an airtight plastic container that also includes a moist napkin. Placing a rack spacer between the napkin and the plates is recommended.

Can the Crystal Formers be used for in situ testing?

Crystal Former material is compatible with in situ diffraction testing, provided that a suitable SBS-plate mount is available. The Crystal Formers are also compatible with the PX Scanner from Oxford Diffraction

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?

Yes, our new SBS High Throughput Crystal Former has been specifically designed for robotic compatibility and lower protein consumption. Please visit our automation FAQs for additional information.

How long does equilibration of the channel take?

Equilibration of ions in the microchannel takes anywhere between 12 hours to approximately 1 week. Smaller salts and buffers will diffuse more rapidly, while larger polymers and macromolecules will take up to a week 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 the 96-condition SuperCOMBI screen in parallel to your regular vapor diffusion screens. You may also use your own vapor diffusion screens in the Crystal Former.

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 in the channels are harvested from the back of the Crystal Former. Use an X-acto knife or a sharp blade to cut the 2 or 3 sealing film tabs of the desired channel and lift one edge of the film up with the blade, then use tweezers to remove the film. 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, samples that are at least 1 week old can be stabilized at approximately 50% of the starting crystallization solution. Crystal appearing in the protein or precipitant inlets can be harvested by cutting and removing the top sealing film of the plate, just like one would do with a sitting drop vapor-diffusion plate.

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 careful 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 can thus significantly alter the crystal size.

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. Users may use small, medium and large MW PEGs to do three parallel grid screens around the initial hit. 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 SuperSMART 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 gentle diffusive mixing of protein and precipitant inside microchannels resulting in gentler 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 the protein phase diagram in a different fashion. 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!

What is the minimum volume of sample per channel in the Original format?

The total volume of the channel is 150nL. While some liquid handling robots are capable of dispensing such small volumes (ECHO, Labcyte), manually set drop sizes of 0.5 µL each for the protein and precipitant are recommended.

What is the minimum volume of sample per channel in the Scale-up format?

The total volume of the channel is 1.1 µL. Manually set drop sizes of 1.5 µL for the protein and 0.5-1.5 µL precipitant are recommended.

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.

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.

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 than 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 the 96-condition SuperCOMBI screen. You may also use your own vapor diffusion screens in the Crystal Former.

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/translate a crystallization hit?

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 alter crystal size.

How do I optimize a hit from PurePEG conditions?

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).