forum
T e c h n i c a l

N o t e sa n d

No. 7, Update 2015
A p p l i c a t i o n sContent
1. 96 Well CrystalQuickTM Plates forHigh-Throughput Vapour Diffusion
Applications		 - CrystalQuickTMSW (Square Wells)		 - CrystalQuickTMRW (Round Wells)		 - CrystalQuickTMLP (Low Profile)

2. LBR Plates for Polarised Light and
UV Light Detection
3. CrystalQuickTM Plus with
Hydrophobic Surface
4. Appendix

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L a b o r a t o r y

W o r kAdvanced High-Throughput Platformsfor Protein Crystallisation

The most widely used method for the determination ofprotein structures is the X-ray analysis of protein crystals.
This type of analysis has led to major advances in basic
research, particularly in the fields of structural genomics, andstructure-based drug design.

In recent years the field of protein crystallisation has
undergone fundamental changes. There has been a dramatic rise in the demand for sophisticated, specialised andflexible platforms for high-throughput crystallography.Thesesystems need to provide multiple screening capabilities,
excellent optical characteristics and be suitable for use
with small sample volumes that may or may not containcompounds that lower the surface tension of the sample.
Microplates designed to be used with these systems have
similarly advanced in both capability and effectiveness.
The architecture of these plates facilitates low protein andreagent consumption whilst maintaining compatibility withhigh-density applications and automated equipment.

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No. 7, Update 2015

Certain unique polymers provide a low birefringent
background (LBR) for crystal inspection under polarised
light. Microplates can also be treated to produce ahydrophobic surface which prevents the deformationof crystallisation drops in the crystallisation wells. Thisis especially important when surface tension reducing
substances, such as detergents, are used whenworking with membrane proteins, which enables theuse of flat bottom crystallisation plates when investigating
membrane proteins.

1. 96 Well CrystalQuickTM Plates for High-Throughput
Vapour Diffusion Applications

cover slide

VIEWsealTM

siliconegreasesampledrop
reagent solutiona) Hanging Dropb) Sitting Drop

Figure 1: Crystallisation methodsa) Hanging Drop b) Sitting Drop

The two vapour diffusion techniques, sitting drop andhanging drop, are the most common methods forcrystallising proteins. (Fig. 1 a, b). Using these methods,
a sample droplet, formed by mixing a protein solution
with a reagent solution, is incubated alongside a larger
volume of the same reagent solution. Both, sample
droplet and larger reagent solution, are sealed within
a single well. The reagent solution can contain a wide
range of chemicals, e.g. buffers, salts and precipitation
agents. When the sample solution is prepared by mixing
this with the protein sample, the concentration of thereagent solution is diluted which initiates a concentration
gradient between the sample solution and larger reagent
solution within the vapour diffusion well. Equilibrium isthen achieved by passive diffusion, and as this process

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occurs, the concentration of proteins and chemicals in
the crystallisation droplet rises. If the correct conditions
have been reached, the process of crystallisation isinduced.
Numerous factors affect the crystallisation of proteins
and in general cannot be predicted. A large numberof experimental conditions often need to be examined
before optimal parameters for crystallisation can bedefined. Due to the large number of conditions that haveto be tested, protein crystallisation still represents a majorbottleneck in the determination of protein structures. The
use of high-throughput equipment and techniques suchas pipetting robots and imaging systems has greatly
reduced the time necessary to define the appropriate
conditions for crystallisation.The Greiner Bio-One rangeof CrystalQuickTM microplates for sitting drop applications
has been specifically designed to be compatible with awide range of automated systems.
The CrystalQuickTM microplates (Fig. 2) have beendeveloped in cooperation with the Genomics Institute ofthe Novartis Research Foundation (GNF) in San Diego(USA), the Max-Planck Institute (MPI) and the Protein
Structure Factory (PSF) in Berlin. The geometry of theFigure 2: CrystalQuickTM plates with different well profiles

plate closely follows that of a standard 96 well microplate.
Each reservoir corresponds to a standard well position
but contains an additional elevated platform that consistsof one or three sample crystallisation wells. The platesforum

No. 7, Update 2015

are optimised for sealing with an adhesive film such as
VIEWsealTM or AMPLIsealTM. All CrystalQuickTM microplates
are available in an LBR version ( chapter 2) for use withpolarised light and partially with a hydrophobic surface( chapter 3).
CrystalQuickTM SW (Square Wells) (Fig. 3)
With three sample crystallisation wells per reservoir,
CrystalQuickTM SW makes it possible to test 288 samples
per plate. Each sample crystallisation well allows for amaximum droplet volume of 4 μl and the flat bottom ofeach well provides good optical clarity.

Figure 4 a): Well profile, Crystal QuickTM RW

Figure 4 b): Protein crystal in CrystalQuick™ RW, the image was kindly
supplied by B. Blattmann, NCCR Structural Biology, Zurich (Switzerland)

Figure 3 a): Well profile, Crystal QuickTM SW

CrystalQuickTM LP (Low Profile) (Fig. 5)
Low profile crystallisation plates simplify crystal harvesting
due to the angled walls of the crystallisation wells. The
low profile of the plates reduces storage requirements
and the flat base of the sample crystallisation wellprovides excellent visual clarity.

Figure 3 b): Crystallisation of lysozyme in Crystal QuickTM SW

CrystalQuickTM RW (Round Wells) (Fig. 4)
With three round crystallisation wells per reservoir,CrystalQuickTM RW allows 288 samples to be tested perplate. The base of the crystallisation wells are concaveand hold a maximum sample droplet volume of 1.9 μl.

Figure 5 a): Well profile, Crystal QuickTM LP

0.5 mmFigure 5 b): Crystallisation of Iysozyme in CrystalQuick™ LP, Robo DesignInternational Inc., Carlsbad (USA)www.gbo.com/bioscience

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2. LBR Plates for Polarised Light and UV-LightDetection
An important issue for high-throughput protein
crystallisation is the visual inspection of the droplets.
Thanks to the birefringent properties of crystalline material,
the application of polarised light in crystal identification isa powerful tool. The use of crossed polarisers in imaging
systems results in light coloured crystals with the majorityof protein crystals. The only drawback with this systemis the impossibility to differentiate between salt crystals
and protein crystals. However, any other non-crystalline
material remains dark. With crystallisation plates being
manufactured out of polystyrene, this produces a highly
coloured background which interferes with crystal
identification. To counteract this unwanted phenomenon Greiner Bio-One developed a unique range of lowbirefringence plates (LBR).

a)b)

In addition to providing low birefringent backgrounds(Fig. 6), the LBR plates also exhibit extremely low
autofluorescence. Low autofluorescence lends itself to1a)

2a)

3a)

c)
1b)

2b)

3b)

Figure 6: CrystalQuickTM plates in polarised light:(a) Standard versions with strong birefringence(b) LBR versions with lower birefringence(1) CrystalQuickTM RW (2) CrystalQuickTM SW (3) CrystalQuickTM LP
LBR white light

crystal detection via UV-light (Fig. 7) as this method relieson the amino acid tryptophan fluorescing when excitedby light in the UV wavelength range. The advantage ofthis method is that salt crystals will not fluoresce thus
reducing any false positives.

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LBR UV-light

PS UV-light

Figure 7: Comparison of absorption in the UV-range (Fig. 7a) and ofautofluorescence (Fig. 7b) of polystyrene and LBR plates. Fig. 7c shows
the performance of polystyrene and LBR plates for the detection of protein
crystals with UV-light. Images are courtesy of PLS-Design, Hamburg(Germany).

For an overview of our LBR plates see Appendix.

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3. CrystalQuickTM Plus with Hydrophobic Surface
Approximately two thirds of drug targets today aremembrane proteins. Structural analysis of membraneproteins is therefore of major interest to biopharmacompanies in their drug development process.
Nevertheless, expression, purification and crystallisationof membrane proteins are regarded as exceptionallydemanding. Even microplates have to meet exacting
standards to be useful for high-throughput crystallisationof membrane proteins. CrystalQuickTM Plus plates with ahydrophobic surface are ideal for use with membraneproteins. The properties of a hydrophobic surface
efficiently counteract the potential spreading of sample
droplets caused by the addition of detergents to theprotein solution (Fig. 8). This is also true for droplets
containing surfactant precipitants such as MPD (Fig. 9).

a) Crystal QuickTM LPa) Crystal QuickTM Plus LPa)

Figure 9: Comparison of a CrystalQuickTM LP and a CrystalQuickTM Plus LP
plate containing drops with MPD as precipitant.

An additional benefit of the surface is the reduction ofthe meniscus of the screening solution in the reservoir.
This avoids contamination via ‘creeping’ of the screening
solution by capillary action from the reservoir into thesample well (Fig. 10).

b)a) Crystal QuickTMb) Crystal QuickTM Plus

Figure 10: CrystalQuickTM Plus plates reduce the risk of contamination via
creeping thanks to a low meniscus in the reservoirs.

Figure 8: A standard polystyrene 96 well sitting drop flat bottom plate (Fig.
8a) and a CrystalQuick™ Plus plate (Fig. 8b) with a hydrophobic surface werecompared for their performance with detergent-containing nanoliter drops.
Images of 100 nl drops containing 50 mM n-Octyl-Glucoside are courtesy ofKarl Harlos, The Wellcome Trust Centre for Human Genetics, Oxford (UK).

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For an overview of our hydrophobic plates see
Appendix.

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No. 7, Update 2015

4. AppendixProducts for High-Throughput CrystallographyCat. No.

Plates for Sitting Drop Vapour Diffusion

609 120

96 well CrystalQuickTM RW (Round Wells)

1040609 101

96 well CrystalQuickTM SW (Square Wells)

1040609 171

96 well CrystalQuickTM LP (Low Profile)2080609 820

96 well CrystalQuick

RW LBR (low birefringent background)

1040609 801

96 well CrystalQuickSW LBR (low birefringent background)

1040609 871

96 well CrystalQuick

LP LBR (low birefringent background)2080609 130

96 well CrystalQuick

Plus SW (hydrophobic surface)

1040609 830

96 well CrystalQuick

Plus SW LBR (low birefringent background, hydrophobic surface)

1040609 180

96 well CrystalQuick

Plus LP (hydrophobic surface)

2080609 890

96 well CrystalQuick X plate, LBR

2080609 895

96 well CrystalQuickTMX plate, LBR, hydrophobic

2080

TMTMTMTMTM
TM

Qty./bagTM

Qty./case656 190

Low profile lid

20200

656 101

Lid

1100676 070

VIEWseal , highly transparent sealer, microencapsulated silicone adhesive

100100676 040

AMPLIseal , highly transparent sealer, acrylate adhesive

100100TMTM

Barcode Labelling
Customer-specific barcode labelling is available on
request for all high-throughput crystallisation plates.

FormatWe place great value on the suitability of our highthroughput crystallisation plates with automated systems.
Therefore all microplates have a footprint conforming tothe ANSI 1-2004 standard.

In-Situ X-Ray Analysis
For in-situ X-ray analysis, Greiner Bio-One offers the96 well CrystalQuickTMX Plate. A unique design and anultra thin-walled well bottom, in combination with superiormaterial properties, render CrystalQuick™X especiallysuitable for in-situ X-ray analysis and crystallography datacollection. CrystalQuick™X is characterised by excellent
optical properties. Crystal harvesting is made easy due toinclined well walls and a small well depth. Besides bright
light microscopy, the plate features the use of polarised
and UV-light for crystal analysis. For more information
about CrystalQuickTMX, please read our Forum no. 13“CrystalQuickTMX: Optimised platform for in-situ
analysis of protein crystals” (F073112).

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Germany (Main office): Greiner Bio-One GmbH, info@de.gbo.com l Austria: Greiner Bio-One GmbH, office@at.gbo.com
Belgium: Greiner Bio-One BVBA/SPRL, info@be.gbo.com l Brazil: Greiner Bio-One Brasil, office@br.gbo.com
China: Greiner Bio-One Suns Co. Ltd., office@cn.gbo.com l France: Greiner Bio-One SAS, infos@fr.gbo.com
Japan: Greiner Bio-One Co. Ltd., info@jp.gbo.com l Netherlands: Greiner Bio-One B.V., info@nl.gbo.com
UK: Greiner Bio-One Ltd., info@uk.gbo.com l USA: Greiner Bio-One North America Inc., info@us.gbo.com

Revision: July 2015 - F073 016Lids / Sealers