Gel Based Workflows
For the separation of complex protein mixtures using 2-D electrophoresis, analysis in the 1st dimension (Isoelectric Focusing (IEF)) is carried out using GE Healthcare IPGphor or Multiphor units and the 2nd dimension (SDS-PAGE) is performed using either Bio-Rad Mini-PROTEAN II gel kit for mini gel experiments or on GE Healthcare ETTAN DALT 12 systems for large (24 cm) gel format experiments. All large format gels are prepared using a NextGen Sciences 2DEoptimizer system which enables highly reproducible homogeneous or gradient gels to be cast in either native or denaturing form. This separation technology is also used in differential protein expression analyses using GE Healthcare DiGE labeling technology. Typically 2D gel based experiments can resolve around 2000 protein spots on a single gel and the IEF step can be optimized using narrow range pH gradients to resolve a similar number of proteins across narrow pH gradients. Comprehensive training and guidance on the use of these technologies are available.
Following the completion of electrophoresis non DiGE gels can be stained with either commassie, silver or fluorescent stains and the gel image captured using a GE Healthcare Typhoon High Performance Laser Scanner. DiGE gels can be scanned immediately after electrophoresis without further processing. Captured gel images are imported into either GE Healthcare DeCyder or NonLinear Dynamics Progenesis SameSpots specialized software packages to detect protein spots, compare gels and determine protein expression differences in the 2D-DiGE workflow. Both packages can compare muliplexed gel experiments and carry out this analysis in a fully automated manner with final manual checking being performed at the end of the analysis, and all data output has statistical significance factors applied. Protein Spots of interest can be easily identified and then tagged for automated excision using the DigiLab Genomic Solutions ProPic spot picking robot.
Once excised from gels protein identification of 1D bands or 2D spots begins with automatic trypsin digestion using a Digilab Genomic Solutions Progest digestion robot resulting in peptide digests which are then analysed by either MALDI or nano ESI mass spectrometer techniques. Our Applied Biosystems 4800 Plus MALDI TOF/TOF Analyzer is used for the mass spectral data acquisition of much of the gel based proteomic workflow samples. Data acquisition and data processing is controlled by Applied Biosystems GPS Explorer software and both TOF-MS spectra and MS-MS spectra on the most abundant peptide ions present in each sample are collected. Once acquired data is processed through GPS Explorer to an in house MASCOT database search for protein identification.