Moredun Proteomic Facility Services
MPF staff are pleased to contribute towards experimental design, protocol development, data analysis, etc. Complete CONFIDENTIALITY can be provided where required.
Contact us to discuss your specific requirements.
Where MPF staff contribute towards your research, please recognise this through appropriate acknowledgement and/or co-authorship of MPF and its staff. Click here for Guidelines.
Services Available
DNA Sequencing
All analyses are performed using a GE Healthcare (formerly Amersham Biosciences) MegaBACE ™-500 capillary DNA sequencer. In combination with “DYEnamic™ ET Dye Terminator Kit” technology, the state-of-the-art confocal scanning laser optics of MegaBACE™ provide improved sensitivity and signal consistency across all 48 capillaries. Read lengths of 700-900bp at 99.4% accuracy are achievable depending on the quality of the template DNA submitted for analysis.
Pre-Fractionation of Complex Protein Mixtures
An important initial step in any proteomics workflow is pre-fractionation of complex protein mixtures, e.g. crude cell lysates, into manageable narrow pI range fractions that facilitate high resolution analysis by subsequent 2-D gel electrophoresis. This can be achieved in a number of ways including liquid iso-electric focusing (liquid IEF) and more traditional ion-exchange chromatographic methods.
2-D Gel Electrophoresis
The MPF houses a number of GE Healthcare IPGPhor™ IEF (1st dimension) and MultiPhor™ and Ettan-DALT™ (2nd dimension) platforms. A wide selection of 1st and 2nd dimension gel formats is available from our centrally held consumables stock ranging from smaller (7cm) gels for pilot experiments to large (24cm) gels for more definitive high-resolution work.
MALDI-ToF Mass Spectrometry
Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-ToF MS) is a highly sensitive technique (femtomole level) that is used primarily for peptide mass profiling and subsequent protein identification via database mining. In a conventional proteomics workflow, MALDI-ToF MS represents the first line approach to downstream analysis and identification of proteins derived from 2-D gels or multi-dimensional chromatographic purification.
Protein Sequencing
N-terminal and internal amino acid sequences of proteins/ peptides transferred to PVDF membranes are determined by automated Edman degradation chemistry using a PE Biosystems 494 cLC protein sequencer. Depending on sample quality, sequences of 5-30 amino acid residues can be obtained from 0.5-20 pmoles of protein where the N-terminal amino acid residue is not chemically blocked.
SELDI-ToF Mass Spectrometry
Surface Enhanced Laser Desorption Ionization Time of Flight Mass Spectrometry (SELDI-ToF MS) is a technique that combines on-chip retentate chromatography with mass spectrometric analysis to facilitate the capture and identification of specific target proteins (biomarkers) from complex biological mixtures. Careful experimental design and sequential use of different surface chemistries allows a variety of analyses to be performed ranging from simple comparative protein profiling experiments to detailed characterisation of specific bio-molecular interactions.
Liquid Chromatography
The MPF provides chromatographic separations on standard, micro-preparative and capillary scales. Dionex UltiMate™ and Beckman-Coulter System-Gold HPLC platforms provide for size exclusion, ion exchange, hydrophobic interaction and reversed phase separations for a range of preparative and analytical applications. The Dionex UltiMate™ HPLC can be operated in 2-dimensional chromatographic protocols to yield highly purified target molecules.
Tandem Mass Spectrometry
The MPF has recently secured funding from SEERAD and SEEL to purchase two new tandem mass spectrometers. The acquisition of Bruker Daltonics Ultraflex II ToF-ToF and Esquire HCT-Ultra Ion Trap mass spectrometers will increase our current range of services to include de novo characterisation of peptides and oligonucleotides, analysis of phosphoproteins and other post-translational modifications and determination of macromolecular ultrastructure. Further details of this facility will be posted on this website at a later date.
Bioinformatics
Underpinning the above facilities are computational biology methods provided at Moredun via the Bioinformatics group.