Our protemics platform is based on 2-D electrophoresis method. It allows visualization and comparison of thousands of proteins originating from very diverse samples. We are implementing all latest state-of-the-art improvements to reproducibility, high quality separation and resolution as well as accurate quantification. Below, you can find a short description of our equipment.
IPGPhor 3 enables fast, reliable and reproducible separation of proteins based on isoelectric point (pI). Up to 12 samples applied to immobilized pH gradient (IPG) gels with length from 7 to 24 cm can be run simultanously. High voltage up to 10.000 V enables faster separation with higher resolution, while Peltier cooling ensures a stable and even temperature. The manifold for IPG gels also allows cup-loading method of applying samples suitable particulary for basic proteins. The machine is operated through a control software, which also records the parameters (voltage, current, volthours) during the run.
SE 600 Ruby is used to run SDS-PAGE analyses on standard gel format 16x14 cm. This format is usually used for standard 1-D SDS-PAGE analyses or for optimization of 2-D electrophoresis procedures (protein extraction protocols, electrophoresis parameters...). Up to 2 gels are run at the same time in one electrophoresis unit. The unit is equipped with heat exchanger connected to a circulatory water bath, while electrophoresis buffer is mixed with magnetic stirrer to ensure homogeneuos temperature. Accessories such as gel casting stands, glass plates, spacers, clamps, cams and combs are required for various applications.
Large format of gels 26x20 cm is used for analyses using regular 2-D electrophoresis or 2-D difference gel electrophoreses (2D-DIGE). This gel format in combination with 24 cm IPG gels enables the maximum quality of separation in both dimensions. Capacity of electrophoresis unit is 6 gels at a time. Built-in pump circulates the buffer, which is cooled through a ceramic heat exchanger connected to a circulatory water bath. Accessories to this unit include gel caster and glass plates (regular of low-fluorescence).
The purpose of this unit is colling of electrophoresis tanks during the runs. Temperature range of the coolant is from -10 to +90 °C, thus the unit can be used also as a water bath for various applications. The pressure inside cooling tubes is limited to 0.32 bar, so that the heat exchangers in electrophoresis units can't suffer damage from high pressure. Convenient connectors are used for quick connection and disconnection of different electrophoresis units to cooling tubes.
ImageScanner III enables high resolution image acquisition of gels stained with visible dyes such as Coomassie Brilliant Blue silver. Gels can be scanned in reflective mode (high quality image of the surface of the gel) or in transparent mode (signal intensity picked up through the whole thickness of the gel, enabling quantification in Coomassie Brilliant Blue stained gels).
To use DIGE technology, an appropriate scanner capable of separate detection of three dyes used to label proteins (Cy2, Cy3 and Cy5), is required. These dyes are used to label three samples, which are subsequently analyzed on the same gel. This approach reduces technical gel-to-gel variation so much, that technical replicates are no longer required.
DIGE Imager is a CCD camera based scanner, which enables high resolution scanning of gels, stained with DIGE Fluors or other frequently used fluorescent dyes such as Deep Purple, Sypro Ruby or ProQ Diamond. Gels are scanned inside the glass plates, therefore chances of contamination or damaged gels is eliminated.
Gel image analysis is performed using dedicated software packages. Classical workflow includes importing and editing gel images, spot detection and quantification, matching of spots between the gels, statistical tests and differential expression analysis. Most operations are fully automated in order to exclude additional bias introduced by user intervention. However, limited manual operations are allowed. After analysis is complete, a set of protein spots can be exported a picklist to the robot to be excised for protein identification.
Protein spots of interes have to be excised from the gel in order to be analyzed. The robot is highly efficient and accurate at spot excision with minimal chances of contamination of gel plugs with keratin. Spot excision is automated using a software-generated picklist, which contains coordinates of designated spots relative to two reference markers glued to glass plates. Picker head is covered with special coating, which prevents cross-contamination. During spot picking, the gel is submerged in water to prevent shrinking. Excised gel plugs are transferred to 96-well microplates, ready for further analysis with mass spectrometry.