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Proteomics

The IFR Proteomics facility advises and collaborates with IFR scientists for the analysis and identification of proteins using 2-Dimensional gel electrophoresis (2DE) and mass spectrometry (MS). Dr Fran Mulholland manages and maintains the IFR proteomics lab and is the first port-of-call for IFR scientists on experimental design, protein extraction procedures and the appropriate protein separation and identification technology to employ.  The IFR lab specialises in gel-based proteomics techniques with well established published protocols. For protein identification the facility has access to the Norwich Research Park-wide Proteomics Facility where there are several mass spectrometers available for proteomic work.

Fran Mulholland manages and maintains the IFR proteomics lab

IFR-based facilities

2DE systems including:

  • 3 IPGphor (GE Health Care) systems capable of running 7-24cm first dimension IPG strips,
  • 3 Investigator second dimension gel casting and running systems for 24 x 22cm gels
  • a2DEoptimizer (Nextgensciences) for casting custom gradient gels,

Pharos FX Plus Molecular Imager (Bio-Rad) for 16 bit fluorescence measurements on a variety of media including gels, blots, microscope slides, and tissue culture plates. It is DIGE capable with 3 excitation lasers and multiple scanning options.  It has a maximum scanning area of 350 x 420 mm at resolutions of up to 50 um.

GS-800 Calibrated densitometer (Bio-Rad) for the optical density analysis of visual stained, e.g silver and coomassie, media.

2D Gel analysis software

  • ProGenesis SameSpots (NonLinear Dynamics)
  • ProteomWeaver (Bio-Rad)
  • PD-Quest (Bio-Rad). 

Quantity One (Bio-Rad) software for lane and band analysis of 1D gels.

Investigator ProPic Protein Picking Workstation for the accurate and precise excision of protein spots from 2-D gels.

Investigator ProGest Protein Digestion Workstation for the enzymatic digestion of proteins contained in pieces of gel after electrophoretic separation. The ProGest performs enzymatic digestion on up to 96 samples simultaneously .

Norwich BioScience Institute based MS facilities

  • The Bruker UltraFlex MALDI-ToF/ToF instrument offers high sensitivity and high resolution, allowing accurate identification of peptides obtained from trypic digests (protein fingeprints). This allows database searching to provide protein identification with high confidence levels. Off-line fractionation and automated spotting of MALDI target plates is established. An additional feature of this machine is its ability to provide some sequence information from intact proteins
  • The Q-ToF 2 (Waters) with a nano-flow liquid chromatography system.
  • The LTQ-Orbitrap (Thermo Fisher Scientific) provides very high mass accuracy typically less than 5 ppm while the fast scan speeds allow great sensitivity. This machine is typically used with a nano-spray and reverse-phase chromatography for the analysis of relatively complex peptide mixtures. This machine is also used for quantitative analysis with SILAC or 15N labelled material or various stable isotope tags, such as iTRAQ.