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Quality control of colorimetrically stained gels and blots

Comparison of 2 methods

Dipl.-Ing. Heiko Mixtacki, Biostep GmbH, Jahnsdorf, Germany

In the pharmaceutical and food industry, colorimetrically stained samples like gels stained with Coomassie® are an important method for quality control. The acquisition of these samples for documentation and quantification purposes can be realised in two different ways.

In addition to conventional camera systems, there are also scanning systems available. Of course, both of them have their applicative orientation and therefore advantages as well as disadvantages. In general, all colorimetrically stained gels, blots and TLC samples can be detected with a camera or scanner–based system without any problems. Both systems use CCD elements for converting the incident light into electrical signals. But this is all for the commonness.

In principle, the geometric shape of the CCD element as well as the adapted optics are different at cameras and scanners. The following table compares the most important parameters.

Technical parameters Camera system Scanner system Notes
Shape of CCD chip Rectangle Row
Optics Zoom objective No objective, micro lenses
in front of each CCD element
Resolution Depending on sample size According to the dpi value  defined,
constant over the complete acquisition area
Advantage for scanner
Speed The complete sample will be
detected with one acquisition
By means of a pecking motor, the CCD row
is transported in dependence on the
defined resolution
Advantage for camera
Calibration with
greyscale wedge
Possible but limited Yes Decisive advantage
for scanner
Dynamic range 8 – 14 Bit 8 – 16 Bit
Exposure time 0.04 sec – 1 sec for
colorimetrically stained samples
Approx. 0.5 sec per row (approx. 1 min for
a gel of 18 x 17cm at a resolution of 300 dpi)
Advantage for camera
Applications Fluorescence, chemiluminescence,
colorimetrically stained samples
Colorimetrically stained samples
Table 1: Comparision between camera and scanner-based systems

Camera-based systems have there biggest advantages in the field of fluorescent and luminescent samples. Camera and scanner based systems are both suitable for the documentation of colorimetrically stained gels. However, the scanner is definitely the better choice for the quantification of these gels – due to the possibility of calibrating the system via greyscale wedge, a higher dynamic range up to 16 Bit as well as a constant resolution of up to 4800 dpi.


Image 1: Scanner system biostep® ViewPix 900 for transmittance and reflectance acquisitions.


Image 1 shows an example for such a laboratory-suited scanning system which is also perfectly dedicated for moist samples (e.g. gels).

Apart from the metrological aspects, the GxP conform data generation and data processing play an important role in the pharmaceutical and food industry. The following requirements must be met:

  • Method-based control of the acquisition systems
  • Record of all system-relevant detection parameters to each sample
  • Hierarchical right system within the user administration
  • Audit trail for the documentation of the changes at the image data
  • Databank-based archivation incl. search functions
  • Complete IQ/OQ qualification of the complete system

The biostep control and documentation software argusX1® offers a comfortable solution for these requirements. The control of the scanner series ViewPix is based on individual, application-dependent methods which are set according to SOPs. This procedure simplifies and automates the operation and guarantees a high reproducibility. The acquisition methods contain the following essential factors:

  • Control of the scanner in transmittance mode (e.g. gels) or reflectance mode (e.g. blots)
  • Acquisition of the samples as 8, 16 Bit greyscale image or 24, 48 Bit coloured image
  • Setting of brightness, contrast, gamma
  • Selection of the colour channel (red, green, blue, user-defined)
  • Orientation of the image (rotate, reflect, invert)
  • Analysis of the over/underexposure

Image 2: Standard mode for scanner control in argusX1®.


The calibration of the scanner for greyscale acquisitions is a very important issue for the GxP conformity. Correspondingly, age-related changes and differences between the systems and application-dependent settings are standardized.

Essential for the daily work is the possibility of a method-based archivation of different calibrations which can be based on different greyscale wedges; because a different greyscale wedge should be used for gels stained with Coomassie® than for gels stained with SilverStain.

Image 3: Professional mode for scanner control in argusX1® (excerpt).

 

Saving the calibration in the method is a temporary process. An existing calibration is going to be replaced by a new one according to SOP regulations e.g once a week at the latest. A subsequent estimation of the parameters which are beyond the calibration function (e.g. quantity of calibrating points, max. deviation, performer, time of calibration) would not be possible anymore. In order to completely document this process, the used calibration with all its parameters must be saved to each sample image.

Image 4 shows an appropriate example.

Image 4: Representation of the complete calibration information to a saved image in the database.

The biostep control and documentation software argusX1 does save information to the type of scanner, acquisition method, calibration, colour optimisation and procession history etc to each sample image. With these detailed data, a subsequent GxP conform review of the acquisition situation can be made.

However, the properly meaning of the calibration will be realised in the analysis software. Together with the image, the calibration saved to each acquisition in argusX1® will directly be transferred to the analysis software for electrophoresis gels Gelix One®. The calculation of the lane and band information in the 1D software is based on OD values when applying the calibration.

Image 5:Analysis software Gelix One® for 1D gels.

 

Summary:


In the pharmaceutical and food industry, using a scanner-based system is more recommended than using a camera-based system for the acquisition, documentation and analysis of colorimetrically stained gels, blots and TLC samples.
The combination of a scanner with a suitable control and analysis software is decisively for completely meeting GxP guidelines.

 

 

Trademarks used:

  • Coomassie® is a registered trademark of Imperial Industries PLC
  • argusX1®, biostep®, Gelix One® are registered trademarks of biostep GmbH
  • Usually, existing protective rights are marked. However, the lack of such signs does not mean that there are no protective rights.

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