Easy set-up for high throughput protein crystallization experiments using the VIAFLO 96 and VIAFLO 384 handheld electronic pipette

  • Easy set-up for high throughput protein crystallization experiments using the VIAFLO 96 and VIAFLO 384 handheld electronic pipette

    How INTEGRA’s 96 channel electronic pipette aids liquid transfers in 96 well plates for sitting drop vapor diffusion crystallization screens

    Protein crystallization and subsequent X-ray analysis have contributed to major advances in the determination of protein structures. Successfully growing protein crystals is challenging and depends on many factors, such as the nature of the reagents, the pH and the temperature. The sitting drop vapor diffusion technique is a protein crystallography method that is very well suited to a 96 well crystallization plate format. All wells of a plate of this type can be filled simultaneously with perfect tip alignment using the VIAFLO 96/384 handheld electronic pipette with a 96 channel pipetting head. Using a single aspiration step reduces the time to cover the entire well plate, which helps to prevent evaporation of precious samples, resulting in more homogeneous crystal growth across the plate.

  • Table of contents

    Protein crystallization and subsequent X-ray analysis have contributed to major advances in the determination of protein structures. Successfully growing protein crystals is challenging and depends on many factors, such as the nature of the reagents, the pH and the temperature. The sitting drop vapor diffusion technique is a protein crystallography method that is very well suited to a 96 well crystallization plate format. All wells of a plate of this type can be filled simultaneously with perfect tip alignment using the VIAFLO 96/384 handheld electronic pipette with a 96 channel pipetting head. Using a single aspiration step reduces the time to cover the entire well plate, which helps to prevent evaporation of precious samples, resulting in more homogeneous crystal growth across the plate.

Key benefits

  • Accommodate and process 96 well crystallization plates on the VIAFLO 96/384 for high throughput crystallization screens.
  • Increased pipetting precision when pipetting into reservoir or crystallization wells.
  • The VIAFLO 96/384 handheld electronic pipette allows easy and rapid set-up of up to 96 different crystallization conditions for increased productivity and reproducibility.
  • Optimal pipetting heights for transfer of proteins and reagents directly into the crystallization plate are guaranteed.
  • The VIAFLO 96/384 allows for fast set-up of crystallization reactions, which helps to avoid evaporation effects, resulting in more robust crystal growth.

Experimental set-up

The following protocol describes a set-up for performing an initial protein crystallization screen using the VIAFLO 96/384 handheld electronic pipette together with a three position stage (Figure 1).

In this protocol, a 96 deep well block filled with 96 different reagents (Morpheus® HT-96, Molecular Dimensions), placed on Position A, is used as the source for a 96 condition crystallography screen. The protein crystallography plate (3 Lens Crystallisation Microplate (UVXPO), SWISSCI) is placed on Position AB, while tips and the tip waste are accommodated on Position B. Three different protein solutions are filled into PCR strips and accommodated on a PCR cooling block, which is exchanged with the Morpheus HT-96 screen plate as required. 

A 96 channel pipetting head (2-50 μl) is used together with 125 μl Sterile, Filter, Low Retention GripTips. Customized VIALINK programs are provided to set up a protein crystallization screen with the VIAFLO 96/384 handheld electronic pipette.

Figure 3: VIAFLO 96/384 with a three position stage.
Three position stage

Downloads: Protocols for automated protein crystallization with VIAFLO 96 and VIAFLO 384

Here you can download the complete protocols used in this application and use them on your VIAFLO 96 and VIAFLO 384. The protocol can be customized with the VIALINK software.

Figure 1: The VIAFLO 96/384 handheld electronic pipette with a three position stage showing the experimental set-up.
Figure 1: The VIAFLO 96/384 handheld electronic pipette with a three position stage showing the experimental set-up. Position A: crystallization screen plate. Position AB: crystallization plate. Position B: 125 μl Sterile, Filter, Low Retention GripTips

Step-by-step procedure:

1. Transfer of the screening agents

Transfer the crystallization screening agents into each well of the crystallization plate

Load tips and launch the program ‘CRYST_SCREEN’ to fill the lens and reservoir wells (Figure 2) of the crystallization plate with the crystallization screening agents. Aspirate 40 μl of the Morpheus HT-96 reagent and dispense 2 μl back into the 96 deep well block (Position A), as indicated in the pipetting program. Dispense 2 μl into each lens well (1-3), and 30 μl into the reservoir well (4), of the crystallization plate on Position AB according to Figure 2. Move the plate slider of the three position stage to access the different wells of the crystallization plate more easily. Discard the pipette tips with the remaining liquid. The accuracy of these pipetting steps is increased by using a pre- and post-dispense.

Tips:

  • Pipetting heights are defined in the program to prevent the tips from touching the bottom of the plate.
  • The volumes dispensed can be easily adjusted to allow investigation of different ratios of protein and precipitant.
Pipetting scheme of the crystallization plate well.
Figure 2: Pipetting scheme of the crystallization plate well. Lens wells: 1-3. Reservoir well: 4.
All four wells (three lens wells and one reservoir well) of the crystallization plate are accessible with the VIAFLO 96/384 handheld electronic pipette.
Figure 3: All four wells (three lens wells and one reservoir well) of the crystallization plate are accessible with the VIAFLO 96/384 handheld electronic pipette.

2. Transfer of protein solutions

Transfer up to three different protein solutions into the wells of the crystallization plate.

30 μl of each of the three different protein solutions are manually distributed into 8 tubes of a PCR strip to allow subsequent aspiration with 8 pipette channels in parallel. The PCR strip containing protein solution 1 is positioned in column 12 of a cooling block, which acts as a support. Instead of loading all 96 tips at once, load 8 tips to the last column of the pipetting head and aspirate 28 μl of protein solution 1. Perform a predispense (2 μl) and then transfer 2 μl to lens well 1 (Figure 2) of each column of the crystallization plate (Figure 4). Perform a post-dispense (2 μl). Discard the tips and exchange the PCR strip for one containing protein solution 2. Transfer the protein solution into lens well 2. Repeat the process for protein solution 3 and lens well 3.

Tips:

  •  Aspiration and dispense speeds can be easily adapted to allow optimized handling of the protein solutions.
  • The column detect mode of the VIAFLO 96/384 handheld electronic pipette offers users haptic feedback for accurate and easier guidance when moving to the next column.
  • Protein solutions can be kept chilled in the cooling block if necessary.
  • Protein solutions can also be stored in a 12 column reagent reservoir if higher volumes of the protein solution are available. The corresponding VIALINK program can be found in the download section.
  • Alternatively, 2 μl of protein solution can be transferred from a PCR strip into the crystallization wells using a VOYAGER 8 channel 12.5 μl adjustable tip spacing electronic pipette with 12.5 μl Sterile, Filter, Low Retention GripTips.
Pipetting and transfer of the protein solution with the VIAFLO 96/384 handheld electronic pipette.
Figure 4: Pipetting and transfer of the protein solution with the VIAFLO 96/384 handheld electronic pipette.

3. Sealing of the crystallization plate

Seal and incubate the crystallization plate.

Tip:

  •  The duration and temperature of sitting drop vapor diffusion depends on your proteins.

Conclusion

  • The VIAFLO 96/384 handheld electronic pipette allows transfer of liquids to crystallization plates in a fast and easy way for high throughput screening of 96 crystallization conditions.
  • Optimal consistency and reproducibility of the results are achieved by using the VIAFLO 96/384 to simultaneously transfer 96 liquids.
  • The protocol for different protein solutions and crystallization screens can be easily adapted using the VIAFLO 96/384.
  • Crystallization plates for sitting drop vapor diffusion in the ANSI/SLAS plate format are optimal for processing with the VIAFLO 96/384 handheld electronic pipette.

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Instruments and accessories

VIAFLO 96/ VIAFLO 384, Handheld Electronic Pipette Three position stage for 96 and 384 well plates 96 Channel Pipetting Head, 50 µl 12 Column Reagent Reservoirs GripTip, 125 µl, Sterile, Filter, Low retention PCR 96 well cooling block CORNING: Corning® Thermowell ® GOLD 0.2 ml 8-Well PCR tube strips SWISSCI: 3 Lens Crystallisation Plate MOLECULAR DIMENSIONS: Morpheus HT-96

VIAFLO 96/ VIAFLO 384, Handheld Electronic Pipette

With this 24, 96 and 384 channel pipette, INTEGRA meets the need for an affordable, compact and easy to use pipette that provides increased productivity. The unique operating concept makes VIAFLO 96/384 as easy to use as any traditional handheld pipette.

Part No. 6001 / 6031

See how it works
The VIAFLO 96/384 is handheld, electronic pipette

Three position stage for 96 and 384 well plates

This accessory expands available stage positions for microplates, reservoirs and tips on a VIAFLO 96 or VIAFLO 384 from 2 to 3 thereby enhancing workflow in a wide range of applications. Beneficially the Three Position Stage also features an indexing function to allow access to 384-well plates using a 96-channel pipetting head.

Part No. 6230

See all plate holder options
Three position stage for 96 and 384 channel pipetting

96 Channel Pipetting Head, 50 µl

Users can simply change the pipetting head of their VIAFLO 96 and VIAFLO 384 to guarantee optimal matching of the available volume range to the application performed. Changing a pipetting head of VIAFLO 96 and VIAFLO 384 is easy and takes less than one minute.

Part No. 6106

Find out more about VIAFLO 96/384 Pipetting Heads Specifications
VIAFLO 96/384 96 channel pipetting head

12 Column Reagent Reservoirs

252 ml , 12 Column Reagent Reservoirs, Pyramid Bottom, Partitioned (21 ml / column), Pre-Sterilized, 25 units, Polypropylene 

Part-Nr. 6362

Find out more about Automation Friendly Reagent Reservoirs
Reservoir for automation with SureFlo™ anti-sealing array

GripTip, 125 µl, Sterile, Filter, Low retention

INTEGRA uses a unique polypropylene blend to mold Low Retention GripTips with heightened hydrophobic properties. Using Low Retention GripTips prevents low surface tension samples from spreading out and “wetting” the inner wall of the tips, allowing them to bead-up for a maximum liquid recovery.

Part No. 6565

Learn more about low retention pipette tips
 Low Retention GripTips are offered in 6000 series racks as non-sterile, sterile and filter options.

PCR 96 well cooling block

Aluminium 96 and 384 well PCR cooling blocks that keep reagents chilled during sample preparation in a PCR plate. The cooling blocks feature a SBS footprint.

Part No. 6250

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PCR cooling block

CORNING: Corning® Thermowell ® GOLD 0.2 ml 8-Well PCR tube strips

  • Tube strips consist of eight 0.2 mL tubes connected together
  • Cap strips are sold separately
  • Made of thin wall polypropylene and designed for precise fit in heat blocks for optimum heat transfer
  • Certified DNase- and RNase-free
  • Autoclavable at 121°C

Source: Website Merck KGaA

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SWISSCI: 3 Lens Crystallisation Plate

The SWISSCI 3 Lens Crystallisation Plate is an ideal product for protein crystallisation presented in a 96-well plate format. The SWISSCI 3 Lens Crystallisation Plate offers unique properties that make it ideal for both nanolitre crystallisation screening and microlitre optimisation alike. Available in optically superior polymers and with an innovative well design allows easy crystal viewing and retrieval.

Source: SWISSCI 3 Lens Crystallisation Plate PDF

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SWISSCI 3 Lens Crystallisation Plate

MOLECULAR DIMENSIONS: Morpheus HT-96

Features of Morpheus®:

  • Simple and effective 3D grid design covering a range of pH, PEGs and salt additives.
  • 49 low molecular weight ligands promote both initial crystal formation and lattice stability.
  • Ideal for membrane proteins (contains PEGS and low salt)
  • Reduced crystal "stress" - all conditions are cryo-protected.
  • Derived from extensive data mining of over 33,000 PDB entries.
  • Available in both 10 mL tube and 96 condition deep well block formats.

Source: Website Molecular Dimensions

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Customer’s voice

This is a very solid reliable machine. Makes cell plating a lot easier with accuracy and reduced strain on arms and shoulders. Also provide flexibility by allowing to swap out heads for different occasions.

Daniel Tsai, NGM Biopharmacueticals inc