This facility offers service and expertise in gel-based proteomics by two-dimensional gel electrophoresis (2-DE). With 2-DE proteins are separated according to two different protein characteristics: 1.) protein charge (isoelectric point, pI) and 2.) molecular mass. Since hundreds to thousands of protein spots can be separated within one 2D gel it is especially suitable for the large-scale screening of complex protein extracts. Here, the separation of intact proteins allows not only the study of protein levels (spot intensity) but also the detection of modifications as well as specific isoforms (spot position).
In addition to 2-DE the facility offers also various native electrophoresis techniques to study protein interactions and complexes:
Two-dimensional gel electrophoresis (2-DE)
Protein patterns of different samples can be compared to investigate e.g.:
- disease mechanisms (including biomarker screening)
- influence of drugs, environmental factors, growth or developmental stages, regulators etc.
- quality of commercial protein extracts (quality control)
Our group has a long term expertise in the analysis of numerous proteomes and subproteomes from bacteria to man to investigate various research questions. Main emphasis is on clinical proteomics to study disease mechanisms with the own research focus on human lung diseases.
Equipment and main approaches
The charge-dependent separation in the first dimension (isoelectric focusing, IEF) is performed with an IPGphor 3 IEF system or a Multiphor II flatbed system (GE Healthcare). Following IEF the proteins are separated in the second dimension (SDS-PAGE) by their molecular mass in an Ettan DALT II system (GE Healthcare) which allows the separation of up to 12 gels.
The protein pattern is visualized by labelling the proteins with fluorescent dyes (e.g. Cy3, Cy5) prior separation (2-D DIGE) or staining the gels with silver nitrate or different fluorescent dyes. Several fluorescent dyes allow also the specific detection of phosphorylated or glycosylated proteins. Since 2D-DIGE offers high accuracy and sensitivity in spot detection and quantification it represents the current major 2-DE approach. The two major DIGE approaches are the minimal DIGE (50 µg protein) and the cysteine-specific saturation DIGE for the analysis of scarce samples (5 µg protein).
After the separation (2D-DIGE) or staining the gels are scanned with an ImageScanner III (GE Healthcare) for silver stained 2-D gels and Octoplus QPLEX (NH DyeAGNOSTICS) or MolecularImager FX Pro (Bio-Rad) for fluorescent 2-D gels. The protein pattern is analyzed with specific softwares, e.g. Delta 2D (Decodon) or Melanie (GeneBio). Interesting protein spots are identified and further characterized in our mass spectrometry facility.
|IEF gel||35 €||55 €|
|2-D gel without staining||70 €||90 €|
|Silver stained 2-D gel||85 €||110 €|
Fluorescent dyes, DIGE kits or Refraction-2D kits will be charged extra.
Dr. Steffen Ohlmeier
Faculty of Biochemistry and Molecular Medicine
University of Oulu
Aapistie 7B (office F009, lab F004)
90230 University of Oulu, Finland
tel. +358-(0)294 481209
e-mail: Steffen.Ohlmeier (at) oulu.fi
Last updated: 28.2.2020