Membrane Protein Laboratory | Equipment available at the MPL

The membrane protein laboratory at Diamond has all the equipment necessary for expression, purification and crystallisation of membrane proteins. All this equipment is available to MPL staff and visitors.

Integrated robotic crystallisation system

Rhombix crystallisation robot in the MPL

The ThermoFisher integrated crystallisation system has a series of robotic components which allow all stages of crystallisation and plate inspection to be completed automatically. The system consists of:

• a Hamilton Robotics STAR liquid handler robot for plate preparation.
• a Digilab/Genomic Solutions Honeybee 16+1 nanodrop system for protein and precipitant drop preparation
• two Abgene plate sealers for PCR and crystallization plate sealing
• ThermoFisher plate hotel and imaging systems holding 720 plates each
• Two VAL robots to move plates between the components
• The system is managed by a database which has information about how a crystallisation plate was prepared and also images of the resulting crystallisation drops.

This system can prepare and image up to 10 crystallisation plates in one go, without user intervention. It can prepare and seal up to 50 screen plates from 96 x 15 ml tubes.

Hamilton Star liquid handler robot

Hamilton Star liquid handler deck.

The MPL Hamilton Robotics Star is a liquid handler with eight channels. It can transfer solutions of volumes ranging from 2ul to 1ml, using disposible tips from 50 ul to 1 ml. It can also use sets of 300ul non-disposible metal tips. We use the Hamilton to transfer well solutions from 96 screen solutions in 15 ml tubes in a rack to crystallisation plates. It is also used for preparing deep-well blocks, transferring solutions from deep well blocks to plates and mixing solutions in plates and deep-well blocks. The Hamilton STAR has an arm for transferring plates to and from the deck. It can also place lids onto plates and remove lids.

DigiLabs Genomic Solutions Honeybee Nanodrop Robot

Digilabs Genomic Solutions Honeybee robot.

The Honeybee 16+1 nanodrop crystallisation robot from DigiLabs Genomic Solutions® takes pre-prepared 96-well crystallisation plates with well solution added to the reservoirs. The system has 16 ceramic tips for transfer of 50 to 200 nanolitres of well solution to the crystallisation stage. In addition it has one separate channel for the protein solution, allowing dispensing of 50 to 200 nl drops of protein solution onto the shelf. This system also has a humidity chamber to prevent the drops from drying out during the plate preparation process.

ThermoFisher Rhombix Cs 750i hotel and imaging systems

Vision hotel and imager	Images of crystals from the Rhombix database © Diamond Light Source Ltd.

The MPL has two ThermoFisher Rhombix Cs750i crystallisation plate hotel and imager systems. Each system takes up to 720 crystallisation plates, both 96-well SBS format and 24-well Linbro plates. The Vision units are kept at 4oC and 20oC. The system can take a number of other plate formats, although we currently only use the Wilden/innovaplate 96 well plates and Linbro plates. Plates can be inserted by hand using the door shown in the image or automatically using the robotic door on the other side of the system. Imaging schedules are available which allow the system to take shots of the crystallisation drops in plates at intervals using polarised and unpolarised light.

Images of crystallisation drops are stored in a database which can be accessed either on the viewing station or on a remote computer. The user can inspect their crystallisation plate images and information about their crystallisation conditions from a PC in their home lab. They can also design new experiments from a remote computer.

Fluidigm microfluidics crystallisation robot

Fluidigm microfluidics system

The Fluidigm® Topaz® crystallization system uses microfluidic chips and a technique called free interface diffusion (FID) for crystallisation of very small quantities of protein. The systems brings together 10 nanolitre samples of protein and crystallization agents in microfluidic capillaries, allowing the two solutions to mix by diffusion. Crystals form as the solutions mix. The experiments are prepared in Topaz screening chips, which take 1 microlitre of protein solution and 96 different crystallization reagents. The plates are prepared using the FID crystalliser ® system, which brings the solutions into contact and seals the capillaries. Samples are then imaged in the AutoInspeX Workstation II ® with the Topaz® database to store the images. Plates are generally imaged over a week. Crystals obtained early in this process are more likely to be protein crystals whereas crystals obtained after several days are more likely to be salt crystals.

PX scanner system for testing crystals in crystallisation plates

PX scanner

The PX scanner from Oxford Diffraction is a compact system for taking both optical and X-ray images of crystallisation plates. The system has a fixed anode X-ray generator with a vertical X-ray beam and a CCD mounted horizontally above the plate position. Crystallisation plates are inserted into a slot and optical images can be taken either for every crystallisation drop in the plate or for drops that are known to have potential crystals. An area of the drop is selected and the plate is moved into position above the X-ray generator. The 200 micron vertical X-ray beam passes through the plate and the crystal and the diffraction pattern is recorded on the horizontal CCD plate above the crystallisation plate.

The crystallisation plate can be rotated by up to 6 degrees and in general several 0.5 or 1 degree rotation images are recorded. These images are processed to remove background diffraction from the plastic of the crystallisation plate.

We have found this system useful for identifying salt crystals. Membrane proteins generally diffract relatively weakly and often it is difficult to obtain diffraction from protein crystals. We are working towards a system on the I24 beamline which will allow us to test diffraction of crystals in situ in crystallisation plates on the beamline. The PX scanner was purchased by the Diamond Light Source as a facility for any visiting users.

If you want to use this system when visiting Diamond, please contact Dr Isabel Moraes(i.moraes@imperial.ac.uk). It is not necessary to make a formal application to the MPL to use this equipment.

Equipment for expression of membrane proteins

Orbital shakers for growth of E.coli and yeast cultures for protein purification

We have three New Brunswick Innova 44R orbital shakers with both universal platforms and platforms for twelve 2 L flasks. These are in room 34 at Diamond. In addition we have a Constant Systems cell disruptor (T series, 1.1 KW 40 psi max) and a Vibra-cell VCX500 sonicator.

In addition we have access to 10 and 50 L bioreactors through the Centre for Structural Biology at Imperial College London in South Kensington.

Purification equipment

Äkta purifier for preparation of membrane proteins

The MPL has two ÄKTA purifiers™ equipped with frac 950 fraction collectors in the cold room:

Model	Max flow rate	Maximum pressure
Äkta purifier UPC 10	10 ml/min	25 MPa
Äkta purifier UPC 100	100 ml/min	10 MPa

Each system measures UV absorption, pH and conductivity. They are controlled by PCs running the UNICORN software. These systems are used for protein purification using columns for affinity chromatography, gel filtration and ion exchange chromatography.

Centrifuges

The MPL has the follow centrifuges available. These can be booked in the diaries in the lab. Anyone wishing to use these centrifuges must first receive training from MPL staff and all use of centrifuges must be recorded in the booking diaries and log books as appropriate.

Centrifuges	Model	Rotor	Tubes	Volumes	Rotor max. speed (rpm)
Ultracentrifuge	Optima L-100 XP	70Ti	Optiseal	8 x 38.5 ml	70000
		45Ti	polycarbonate bottles	6 x 64 ml	45000
Floor standing	Avanti J-26 XPI	JLA-8.1000	bottles	6 x 1 litre	8000
		JLA-16.250	bottles	6 x 250 ml	16000
		JA-25.50		5 x 50 ml	25000
		AllSpin JS-5.2 swingout rotor	microplates, deep well blocks, 15 and 50 ml falcon tubes		5300
Benchtop ultracentrifuge	Optima MAX	MLA-130	Polyallomer Quick-Seal tubes		130000
		TLA-120.1		14 x 0.5 ml	120000
Benchtop, refrigerated	Allegra X-22R	SX4250 Swinging bucket rotor	microplates, deep well blocks, 15 ml and 50 ml falcon tubes		3353
Microfuge	22R	365630	1.5 ml tubes	24 x 2 ml
Microfuge (in cold room)	18		1.5 ml tubes	24 x 2 ml	14000

 

Spectramax M2e microplate reader

Spectromax® M2e microplate reader

The Spectromax ® M2e microplate readerfrom Molecular Devices is a dual-monochromator microplate reader with an additional cuvette port. It can read microplates with between 6 and 384 wells. In general we use the system for 96-well SBS format plates and with cuvettes. The system can read absorbance in the UV and visible wavelengths. It can also measure fluorescence intensity. We use this system for working with membrane proteins fused to green fluorescent protein (GFP) and for measuring protein concentrations.

Fujifilm LAS3000 gel documentation system from Raytek

Fujifilm LAS3000 gel documentation system

The MPL has a

Fujifilm LAS3000 gel documentation system

from

Raytek Scientific Ltd

. This system is used for imaging DNA and protein gels. In addition it is used for in-gel fluoresence of fluorescent labelled proteins tagged with GFP.