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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: info@aviso-ms.de

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.