Electrophoresis Band Quantification - An Art or A Science.

There are so many things to consider when trying to accurately quantify and compare
bands in an electrophoresis gel that it has often been described as more of an art than a
science. The purpose of this article is to identify and discuss the relevant points so that
scientists may be sure that the method of quantifying band material they are using or
intend to use is as accurate as is feasible. Electrophoresis is a powerful, useful and widely
used technique, but is nevertheless fraught with difficulties. The following points include
some of the things that need to be considered for the accurate quantifying of bands. The
discussions on each point are intentionally general and specific reference to any particular
electrophoresis techniques or products has been avoided. This has the effect making the
process appear over complicated. However, once the elements that relate to particular
applications are identified electrophoresis band quantification can be shown to be a
relatively simple science.

1 Good raw material

In order to keep margins of error to a minimum the gels should be run such that the lanes
are well separated and the bands in each lane are as well separated as possible. Merged
bands always introduce inaccuracies and contrary to the belief of some; there is no band
deconvolution mathematics that will accurately separate all types of bands in all types of
gels. In cases where a large number of bands are merged together over variable
background within the lanes, then there can be a real problem in determining what is
background and what is band material. In such cases the margins of error (see point 8)
can be very large. Currently the only methods of solving such problems are to resort to 2D
electrophoresis or to run specially structured gradient gels that are able to cause a better
separation of the bands.

2 Suitable visualization methods

Band staining or labeling for the purposes of visualization is often limited in range and is
frequently nonlinear with regard to the amount of band material and the intensity of the
stain (or other visualization method). This gives rise to two points of consideration. The
first is the fact that accuracy of determining the quantity of material in bands is limited to
those bands that are not oversatuated or overexposed. The second point is that it is
frequently necessary to have some kind of quantity calibration standard within the gel (i.e.
a number of bands of known quantity that will allow the production of standard curves
using a quantity calibration method).

3 Suitable data capture

In order to analyze the bands on an electrophoresis gel the data within the gel must be
acquired for analysis. In the past, densitometers were used to scan and analyze the
electrophoresis tracks. Although optically very good, the old track scanning densitometers
had a number of limitations. They were slow and awkward to use, were unable to handle
distorted gels easily, could give wrong results when bands were not uniform and were

generally limiting in the range of analyses that could be performed. For these reasons it is
currently generally preferred to use an instrument that is capable of acquiring a gel image
that can then be analyzed using image analysis techniques.
The type of image capture device that is used will depend on the kind of visualization
method that is employed. For example DNA / ethidium bromide gels visualized with UV
light will often be captured for analysis using a CCD Video camera system. Images of
radiolabelled gels or blots may be obtained using a Phosphor imaging device or a direct
radiation imager. Similarly, fluorescence or chemi-luminescence visualization methods
may make use of photosensitive instruments designed to produce images from such gels
or blots. In recent years the use of commercial scanners has become a popular method of
acquiring images from stained gels, blots or autoradiograms.
Whichever system is used it is important that the calibration method, if any, is known and
understood. Despite the claims of manufacturers there are very few image capture
systems that are innately linear in response. Those devices that are demonstrated to
produce a linear response are often calibrated to provide that response. This is fine if you
are using the machine in precisely the same way as the manufacturer intended. Any
deviation may require an alternative calibration method unless the demands on the
analysis are not strict and therefore do not require calibration. Also it is often not
necessary to calibrate for non-linearity if alternative quantity calibration methods are used
whereby a series of spots or bands of known value are used to produce a calibration
curve.
Traditionally many machines calibrate transmission readings to Optical Density. As light
passes through an object the degree of absorption is logarithmically related to the density
of the object. Generally the following calculation is used to provide a linear response: Optical Density = -log10[(255-raw pixel value)/255]
New ISO Transmission Density Standards used during the calibration of instruments
designed to examine the density of photographic products with transmitted light make use
of diffuse density which, in effect recognizes the use of diffusers in densitometers. Optical
Density values differ from diffuse density values by a simple multiple of around 1.414.
However, this is only important for those few applications that require absolute values.
Most electrophoresis densitometry applications are examining relative values rather than
absolute values, in which case the difference is academic.

4 Sensible Analytical Methods

Obviously the software used to analyze
the gel image should employ methods that
are accurate and reproducible. It should
also display graphics such that the
scientist is constantly aware of the degree
of accuracy or inaccuracy of the analytical
processes. For example background
subtraction should be displayed on the
lane profile and the band limits (band
edges) should be shown clearly on both
the profile and the lane image.
Furthermore the lane image should be
displayed in alignment with the profile.
Without these simple facilities there is no
real way of visually checking the accuracy
of the analysis.

5 Suitable Calibration Mathematics

In electrophoresis and TLC there are several things to consider when deciding on the
calibration method. The first question relates to whether the visualization method (e.g.
band staining) is significantly non-linear. If it is then a quantity calibration method should
be employed. That is to say a set of dilutions, or a number of bands of known quantity
should be used to construct a quantity calibration curve. This will have the effect of
overriding any intensity calibration. If the visualization method is linear within the range of
the bands of interest, then either quantity calibration or intensity calibration could be used
to overcome any non-linearity of image capture. In some cases intensity calibration can be
done with a singe button press, which invokes the calibration mathematics. However, this
should only be used when no post scanning adjustments have been made to the image.
Unfortunately not all scanners distinguish very clearly between pre-scan and post
scanning adjustments. If post-scanning adjustments are likely, then the gel should be
scanned together with a calibration strip with an incremental series of optical density, or
grey level steps. This calibration strip can then be used to calibrate the image to obtain a
linearity of grey levels or optical density units in the image.
Finally, it is necessary to consider whether the different classes of spots or bands are likely
to stain differently to each other. This is a very common problem in TLC plates and some
protein gels. In such case several calibration curves may be necessary, one for each class
of band. Commonly a dilution series of a standard lane is used. Several band specific
calibration curves are then produced so that the values of corresponding bands of
unknown quantity can be automatically read from the associated standard curve.

6 Suitable Normalization Mathematics

A variety of methods can be employed to normalize against experimental variation
between lanes.
x Band Percentage: The value of each band is expressed as a percentage of the sum of
all the bands in the lane.

x Lane Percentage: The value of each band is expressed as a percentage of the sum of
all the material in the lane including inter band material.
x Lane normalization: Each lane is given an overall value and each band is expressed as
a proportion of that value.
x Normalization to an individual band: A matched band across all the lanes (one that is
deemed to be theoretically unchanging) is given a value and the values of all the other
bands in each lane are adjusted in accordance to the matched band having the given
value.

7 Quality Reporting
Naturally all data must be reported in some way and it should be a simple matter to
present the image, data, associated graphics and calibration methods within a report. For
some applications automated reporting within the analytical software is desirable,
especially when considering the requirements of Good Laboratory Practice. In addition it
should be possible to use copy and paste methods so that scientists can structure their
own reports in the way they find most suitable for their applications.

8 Reproducibility and Accuracy (Error Limits)

All scientific data can be said to have limits with regard to its accuracy. It is therefore
frequently desirable to be able to identify the limits, or margins of error. In order to do this
properly, it may be necessary to analyze each gel several times using different analytical
criteria. For example different forms of background subtraction may be compared, or
varying degrees of band edge determination could be examined. The analytical software
should be capable of allowing this to be done with minimal effort. By pasting a table of
results into a spreadsheet such as Microsofts Excel, changing background subtraction
and pasting the new results into Excel, it is a simple matter to subtract on set of results
from the other and thereby identify the precise effect of altering the background subtraction
method.
Other methods of identifying areas of inaccuracy involve comparing different methods of
expressing the results. Good quality software should contain tables and graphics that
compare and contrast the different measurement fields. For example a scientist may wish
to compare Raw Volume, Calibrated Volume, Band Percentage (calibrated or not
calibrated) and Peak Height. A visual and graphical comparison of these fields will help
identify any problem areas (e.g. when band percentage is not a suitable method for
normalization).

9 Resolution
Sometimes, with certain types of gels containing many merged bands over an uncertain
level of background, it will be found that a scientific determination of error limits produces
margins that are unacceptably broad. In such cases it is tempting to desire equipment with
higher resolution. Generally this is not the answer because improved resolution will often
only help a little, not enough to overcome the unacceptably broad margins of error. For
those applications where a high resolution is necessary and desirable, the scientist should
be very careful of misquoted figures. Many scanners have very tempting quoted levels of

resolution in dots per inch (dpi) far in excess of scientists needs. However these figures
do not account for any averaging between pixels. As a general rule of thumb for
electrophoresis applications scanning above 400 dpi does not tend to improve the
resolution sufficiently to reduce margins of error significantly. Imaging laser densitometers
have a similar resolution (80-90Pm). The resolution of CCD camera systems is a function
of the number of light sensitive points on the CCD chip and the size of the area being
photographed. At best this results in a similar resolution to standard scanners. Therefore
the only way to significantly improve resolution is to purchase a microdensitometer, a drum
scanner, or a high resolution CCD camera system. Whether such instruments would help
is dependent on the application.

10 Speed and Simplicity

On reading all of the above, it might seem that the process of accurately quantifying band
material is really quite complicated. The analytical software should therefore be designed
such that the process becomes as easy and as obvious as possible. This is where there is
a real art in designing software to be highly flexible and address all the possible difficulties
of analysis whilst making it easy to use, obvious and intuitive.
Naturally at Phoretix we believe the most important thing is to have high quality analysis
software. Phoretix 1D software has been designed to address all of the above points.

Dr Bruce Venning, Phoretix International

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