ADVERTISING FEATURE
APPLICATION NOTES
Isolation of protein spots from a silver-stained 2-D gel using the cell separation robot CellCelector™
Automated isolation of protein spots from a 2-D gel
In proteomics laboratories 2-D gel electrophoresis is one of the
most prevalent analytical techniques. In combination with mass
spectrometry it is used for identification of proteins in proteome
analysis. Separated proteins can be stained in the gel by silver
nitrate or colloidal coomassie brilliant blue. For mass
spectrometric analysis appropriate stained spots [1] have to be
excised from the gel.
Figure 2: Screenshot of the CellCelector™‘s control and imaging software, a: Picking list and automatically documented snapshots before and after picking of spots, b: 2-D gel on live image with hole where the spot was excised
Figure 1: a: Assembly of AVISO’s CellCelector™, robot for automated cell harvest, b: Scrape tool, harvest tool for adherent cells and objects from semi-solid or solid media
Technology
The CellCelector™ (fig. 1a) is a robotic system for automated cell
harvest. This patented system consists of an inverted microscope
(1) equipped with a motorized stage (2) and a CCD camera (3),
an exchangeable robotic arm (4) as main functional tool and a
deck tray for disposable tips (5), capillaries and destination plates
(6). The imaging software for detection and picking is a
combination of CellD (Olympus) and AVISO software (fig. 2).
Detection of objects is enabled by predefined spectral and
dimensional parameters. After the specimen on the stage is
scanned, picking and documentation can be done automatically,
based on the generated particle list. Images for documentation
are acquired in high resolution. The harvest tools provide the
collection of adherent or suspension cells as well as colonies in
semi-solid media via mechanically detachment and aspiration.
Further applications such as spot picking are also possible.
Special polished metal capillaries (fig. 1b) are used to scrape off
adherent cells via a crosswise movement of the motorized stage.
The scrape tool is also suitable for picking objects from semi-solid
or solid media and gels. The picking parameters can be fine-
tuned for the users special application.
1b
1a
For proteomics research 2-D gel electrophoresis is the basic technique to separate proteins by means of
molecular weight and isoelectric point. For further analysis of the separated proteins by mass
spectrometry defined spots are excised from the polyacrylamide gel. The CellCelector™ is a novel
automated cell transfer system. Besides the detection and separation of cells, here we demonstrate the
applicability of the CellCelector™ for the excision of stained protein spots from a 2-D gel.
2a 2b
i
Katharina Zoldan, Kathleen Spier, Verena Veneruso, Jörg Lehmann
Fraunhofer-Institute for Cell Therapy and Immunology, Perlickstr. 1, D-04103 Leipzig, Germany
Cell Technology / GLP unit; For correspondence please contact: [email protected]
ADVERTISING FEATURE
APPLICATION NOTES
For this application, a piece of 1 mm thickness is cut from a
silver-stained 2-D gel (fig. 3a) and transferred into a suitable
container on the CellCelector™’s motorized stage. The gel can
be scanned to obtain an overview image. A good contrast is
achieved with bright-field microscopy (fig. 3b). Appropriate
thresholds for the gray value range of the spots are determined
by the user. In a further scan the spots are detected (fig. 3c).
After detection a particle list is generated containing localization
and dimensions of the spots. The user can modify the list by
applying different filters and create a pick project. If accurate
detection is not possible due to fused or irregular shaped spots
the positions can be defined and added to a pick list by clicking
on the live image. All spots in the list will be picked
automatically. A small piece is precisely excised from the center
of the spot (fig. 3a (red box) and fig. 4), aspirated in the
capillary and transferred into a destination well. For every spot a
new capillary can be used. Metal capillaries of different
diameters such as 0.8 mm or 1 mm are available. Since the
CellCelector™ is usually applied to isolate cells a sterile
environment is provided during the picking process. Thus,
contamination of the protein in the spots is prevented. In one
run up to 192 spots can be picked. Images before and after
picking are taken automatically and the location of the excised
spot in the 96-well destination plate will appear in the pick list.
The separated spot pieces in the destination plate are useable
for further treatment (fig. 5). Successful transfer of the spot
piece is assured with a probability of about 90 %.
ii
Figure 3: Different views of a 2-D gel, a: Image of gel piece cut from a 2-D gel; some picked spots are marked with the red box. b: Overview image of a scan with the CellCelector™; protein spots appear in good contrast with gray value imaging. c: Detections made by the software; green marked areas are recognized as particles and will appear in the particle list.
3a 3b 3c
Figure 5: Excised spot pieces in wells of a 96-well destination plate, a:photograph of the destination well plate, side view; b: Scan of a destination well with the CellCelector™, top view
5a 5b
Figure 4: Automatically documented pictures during the picking process, Left: Before picking, Right: After picking, Scale bars = 500 µm
Conclusion
The CellCelector™ is assembled to function as a cell transfer
system but furthermore, it has the basic technical and software
requirements for other applications, too. For instance, as a
multifunctional picking tool it can be used to pick objects from
different media such as semi-solid agar- or methylcellulose-
based culture media or even polyacrylamide gels. Thus the
CellCelector™ represents a useful tool also in fields appart from
cell culture technology such as proteomics. Although the system
is not fully adapted for high-throughput spot picking so far, it
may replace the tedious task of cutting small single spots
manually. The picking and depositing parameters should be
adaptable to the thickness and consistence of the individual gel.
Since the CellCelector™ possesses a fluorescence illumination
unit it should also be possible to enable fluorescence detection
of spots from DIGE gels.
References
1. Richert S, Luche S, Chevallet M, Van Dorsselaer A, Leize-Wagner E, Rabilloud T: About the mechanism of interference of silver staining with peptide mass spectrometry. Proteomics2004, 4: 909-916.
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