Mission Statement

In close collaboration with investigators, the Separations Technology Core provides sample preparation and separation solutions to achieve the highest quality of proteomics expression data possible. This is accomplished by seamless interaction with the other Proteomics Section Cores (Mass Spectrometry and Proteomics Bioinformatics) utilizing the highest degree of professional expert analyses and state-of-the-art technology. The Separations Technology Core ensures quality through the use of automated robotics, high-performance bioinformatics with robust statistical menus, and cutting-edge chemistries. In addition, this Core is engaged in developing new separations chemistries and instrumentation in order to address the detection and quantification of low abundance proteins.

Services Available

• HPLC sample fractionation (HIC—proteins; RP—peptides)
• Preparative IEF fractionation
• Sub-cellular fractionation
• IEF, 1D, and 2D gel electrophoresis with Coomassie or fluorescence staining (Sypro-Ruby for total protein or Pro-Q Diamond for phosphorylated proteins)
• Image analysis of stained protein spots in gels
• Robotic protein spot picking from gels
• Robotic protein digestion, processing, and MALDI target application
• Training in specific applications of separations technology

Quality Control

All fractionation steps and separation media undergo extensive validation in order to ensure reproducibility. Internal standards are employed wherever possible. All quantification chemistries and instruments are certified to produce accurate and precise values. We apply sophisticated statistical tools, available in the Proteomics Bioinformatics Core to evaluate the quality of our data. Protocols and methods are shared with investigators.

Major Core Equipment

• Beckman Optima TLX Table-top Ultracentrifuge with swinging bucket and fixed-angle rotors
• Applied Biosystems 270A-HT Capillary Electrophoresis System
• Protein ProFiler™ Liquid Two Dimensional Electrophoresis System (In Development)

• Invitrogen ZOOM ® IEF Fractionator for preparative IEF
• Nonlinear Progenesis SameSpots with Statistics Toolbox 2D analysis software

• Agilent 1100 HPLC with diode-array detector
• Multiple 1D and 2D gel electrophoresis apparatus
• Digilab Genomic Solutions Pro-Pic II (robotic gel spot picker)
• Digilab Genomic Solutions Pro-Prep (robotic gel plug digestion)
• Fuji FLA-5100 Fluorescence Scanner
• Perkin-Elmer ProEXPRESS 2D Fluorescence Scanner
• Perseptive BioCad Protein Fractionator
• Bio-Rad BioPlex multiplex cytokine analyzer

Protocols

Isoelectric Focusing (2D gels)

SDS PAGE (2D gels)

Fluorescence Staining

Gel imaging

Image Analysis (Nonlinear Progenesis)

Extracting Nuclei