Services - Proteomics

The facility has the complete Proteome Works system (BioRad) which can be used to perform two dimensional separation of complex protein extracts followed by imaging, analysis and spotcutting for eventual identification of the protein spots by mass spectrometry.

First Dimension

The first dimension utilizes an isoelectric focusing device (Protean IEF cell) that can focus 1 to 12 strips simultaneously. The ReadyStrip IPG strips are available in 24 or 11cm lengths with pH ranges of: 3-10, 7-10, 5-8, and 3-6.

Second Dimension

The second dimension utilizes one of two different SDS-PAGE systems: Criterion or Protean II Plus. The Criterion Precast gradient gels accept the 11cm IPG strips while the larger Protean II Plus single concentration gels accept the 24cm IPG strips.

Blotting

In addition the facility has a Trans-Blot Cell for transferring the proteins from a Criterion gel to a PVDF membrane or nitrocellulose.

Staining

The gels can be stained with Bio-Safe Coomassie or SYPRO Ruby. Both stains are compatible with mass spectrometry.

Imaging

The gels will be imaged with the VersaDoc System which utilitzes a CCD camera to capture white light or UV-fluoresence images of the gels.

Analysis

The analysis will be done with the software PDQuest which allows gels to be compared to each other as well as averaged in order to minimize experimental variability. The software can determine which protein spots have changed in concentration and location, and controls the spot cutter. Analysis can be done by the PMGF staff or by the customer at the Facility.

Spot Cutting

The Protean 2-D spotcutter is a robotic platform that images the gel, and then precisely removes the protein spots with a hollow-bit drill attached to a robotic arm for eventual protein identification.

Mass Spectrometry

Usually the cores from the gels are sent to the CCIC- Mass Spectrometry Facility at OSU for protein identification by various mass spectrometry techniques, but they may be sent elsewhere for analysis.

References for research done at PMGF:

  1. Calikowski, T. et al. 2003. J. Cell. Biochem. 90: 361-378. [pdf]

 

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