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The Astbury Centre for Structural Molecular Biology

The JIF Centre for Biomolecular Interactions

Facility Manager: Dr Iain Manfield (i.manfield@leeds.ac.uk) Tel: 0113 343 7279

Contents:

  1. Location
  2. Facilities
  3. Costs
  4. Booking
  5. Instrumentation
  6. Other Facilities

1. Location

The Astbury Centre for Biomolecular Interactions is located in Laboratory 9.108 (Level 9) of the Astbury Building, University of Leeds. For the location of the Astbury Building, see the campus map.

2. Facilities

The Centre presently offers the following services:

  1. Sample analysis using Mass Spectrometry (see Alison Ashcroft's web link) and hplc / fplc.
  2. Sample purification using hplc and fplc.
  3. Oligonucleotide synthesis.
  4. Peptide synthesis.
  5. Amino acid analysis.
  6. Analytical ultracentrifugation (see weblink)
  7. Surface Plasmon Resonance Spectroscopy.
  8. Stopped Flow Fluorimetry and CD.
  9. Microcalorimetry.

3. Costs

There are charges to use the equipment in the centre to cover some consumables, replacement parts and to pay for service contracts. For current charges and charges to use for grant applications, see the FBS Research Support Office web-pages for small and local research facilities; (http://www.fbs.leeds.ac.uk/intranet/RSO/documents/SRFsummarysheetcurrentandapplicationsFebruary2008.xls ; http://www.fbs.leeds.ac.uk/intranet/RSO/documents/6LocalResearchFacilitiescurrentcostingsheetAug08.xls).
Contact Iain Manfield for more information or to discuss your requirements. Rates for non-University of Leeds users will be different – please enquire for details.

4. Booking

To book a facility, prospective users should first discuss their requirements with the Facility Manager and come in person to complete a booking form. Initially, users will be required to provide a charge account number for costing purposes.

5. Instrumentation

High Pressure Liquid Chromatography (HPLC)

Dionex BioLC

The Centre has two Dionex BioLC instruements. One of these instruments is equipped with a PDA-100 Photodiode Array Detector capable of scanning the entire wavelength range of 190-800 nm and the second BioLC is equipped with a ED50 Electrochemical detector that offers pulsed amperometric detection. These instruments are optimally setup for ion exchange separations. The inert, metal-free polyetheretherketone (PEEK) flow path eliminates concerns about biocompatibility and corrosion, allowing users the freedom to choose the best buffers required for their separations. BioLC major applications are in the analysis of:

Dionex BioLC Dionex BioLC

Amino acid analysis

This is carried out using the Dionex AAA-Direct Amino Acid Analysis System specifically designed to separate a wide range of amino acids by gradient anion exchange with Pulsed Electrochemical Detection (PED). No sample derivatisation is necessary.

Dionex Summit

Ideally setup for reverse phase chromatography (RPC) of proteins, peptides and oligonucleotides. The Dionex Summit HPLC allows high sample throughput due to short injection times with the ASI-100 autosampler and a very small volume of the P860 pump. The sample tray is sector divided so that several applications can be set up at one time. It can accomodate analytical and semi-preparative vials. It is equipped with Chromeleon software for dynamic data processing and evaluation as well as photodiode and fluorescence detector systems.

Dionex Summit Dionex Summit

Fast Performance Liquid Chromatography (FPLC)

AKTA Explorer 100 Air

This chromatography system can be used for the purification of proteins, peptides and nucleic acids. It uses one working platform for all techniques and all sample loads from microgram to gram scale. Systems can be selected to use flow rates up to 100 ml/min (pressure 10 mPa ) or up to 10 ml/min (pressure 25 mPa ), ensuring optimum running conditions for all modern high performance media. The UNICORN software control system allows maximum automation and flexibility. The AKTA explorer 100 air is capable of monitoring and excluding air infiltration at the sample and buffer inlets. It has fast method and process development and scale-up for proteins, peptides and nucleic acids.

AKTA Explorer 100 Air AKTA Explorer 100 Air

Applied Biosystems Vision (BIOCAD)

The VISION workstation is designed for analytical or preparative scale automated perfusion chromatographic purification of nucleic acids or proteins. Its systematic method development optimises conditions for an application by identifying variables that affect an application, systematically changing those variables, observing the effects, and using the information to optimize chromatographic conditions. VISION is equipped with MS Windows based software with full process capability from sample injection through fraction collection and analysis including column washing and regeneration.

BioCAD Vision BioCAD Vision

Solid Phase Peptide Sysnthesis (SPPS)

Currently, there is a growing demand for synthetic peptides in the area of vaccine development, anticancer drugs, aggregation inhibitors as well as other applications.

SPPS is quick and easy approach to synthesizing peptides and small proteins. The C-terminal amino acid is attached to a cross-linked polystyrene resin via an acid labile bond with a linker molecule. This resin is insoluble in the solvents used for synthesis, making it relatively simple and fast to wash away excess reagents and by-products. The N-terminus is protected with the Fmoc group, which is stable in acid, but removable by base. Any side chain functional groups are protected with base stable, acid labile groups.

Solid Phase Peptide Sysnthesis

The centre has two peptide synthesizers; an Applied Biosystems 433A and an Advanced Chemtech APEX 396 Multiple Peptide Synthesiser. Both instruments are fully automated and programmable for continuous-flow solid phase peptide synthesis. Although synthesis of peptides up to 100 amino acids in length is possible in principle, the effective length is up to 50 amino acids and depends on the complexity of the peptide.

Apex 396 Apex 396

Oligonucleotide Synthesis

Currently, synthetic deoxyribo - and ribo -nucleotides have wide applications in molecular biology. The centre has several automated solid phase instruments in service for the custom synthesis of DNA and RNA oligomers One instrument is specifically modified for the 2'-ACE chemistry. Solid phase oligonucleotide is conducted with the growing oligo chain attached to a solid support made up of controlled-pore-glass (CPG) beads. The nucleoside is bound to the CPG through a linker attached at the 3'-OH. The 5'-OH is blocked with a dimethoxytrityl (DMT) group, removal of which followed by coupling with a phosphoramidite extends the chain.

Oligonucleotide synthesis

The Dharmacon 2'-ACE RNA synthesis is based on a 5'-OH silyl group protection in combination with an acid labile orthoester protecting group on the 2'-OH.

Specialty oligonucleotides which include modified bases and spacers, modified 5' and 3' ends, as well as modified phosphate backbone can be synthesized on request.

ABI 392 ABI 392 synthesiser

Microplate Fluorescence Reader

The POLARstar OPTIMA is a multifunctional microplate reader that can perform a wide variety of applications for fluorescence intensity, time-resolved fluorescence, absorbance and luminescence as well as fluorescence polarization. The versatile optical system allows switching from top to bottom optics and changing between different measurement principles. By using a xenon flash-lamp and liquid-filled light guides, the instrument can achieve a wide wavelength range. The instrument has a built-in incubator and can be configured with reagent injectors.

polarstar Polarstar Plate Reader

Surface Plasmon Resonance

BIACORE 3000 is a system for real-time biomolecular interaction analysis (BIA) using surface plasmon resonance (SPR) technology. SPR is a non-invasive optical measuring technique that measures the mass concentration of biomolecules in close proximity to a specially prepared surface. Real-time BIA can be used for studying the interactions of proteins, protein conjugates, nucleic acids, lipid micelles and even larger particles such as viruses and whole cells.

BIOCORE3000 BiaCORE 3000 SPR

Stopped-flow Spectroscopy

One of the most frequently used kinetic techniques is stopped-flow. The Centre has three Stopped-Flow instruments in service. The Biologic MOS-450 Stopped-Flow spectrometer (pictured below) has absorbance, fluorescence, fluorescence anisotropy and circular dichroism modes. It is fully software controlled for acquisition and data analysis.

MOS-450 Stopped flow machines

Microcalorimetry

Microcalorimetry is now the biophysical method of choice for non-invasive and non-destructive analysis of biomolecular stability and interactions. The Centre is equipped with differential scanning calorimeters (DSC) used mainly for the study of thermal transitions in dilute solution (protein folding, DNA melting, lipid bilayer transitions, etc) and isothermal titration calorimeters (ITC) used for binding studies in dilute solution (enzyme- ligand , protein-protein, protein-nucleic acid, and/or small molecule interactions).

DSC and ITC DSC and ITC Machines

Other Facilities the Centre offers include:

HETO LYOPRO 6000 Freeze Drier with ice condenser capable of attaining -90 °C for efficient freeze drying of aqueous samples.

Heto HETO LyoPro 6000 Freeze Dryer

THERMOSAVANT SPEEDIVAC for fast and efficient solvent evaporation. This can be achieved at low, medium or high temperatures depending on the sample requirements.

Savant Savant Speedivac

GENESYS UV-Visible scanning Spectrophotometer (covering 190-1100 nm) with a built-in UV and visible emission lines for calibration and verifying wavelength accuracy. The spectrometer has a 6-position automatic cell holder (with one position reserved to hold blank and five sample positions).

GeneSys GeneSys spectrophotometer