Densitometric Image Analysis Software

Software description

The "Densitometric Image Analysis Software" is a software tool to quantify electrophoretic bands in autoradiographs as well as in gels and phosphorimages. Moreover, it allows to determine stepwise equilibrium constants from Electrophoretic Mobility Shift Assays (EMSAs) with accuracies of about 20% instead of the usual factor of about 2.

To validate the software, it has been extensively applied in the following papers:

  • N. Song, T. Nguyen Duc, L. van Oeffelen, S. Muyldermans, E. Peeters and D. Charlier, ‘Expanded target and cofactor repertoire for the transcriptional activator LysM from Sulfolobus’, Nucleic Acids Research, vol. 41(5), Jan. 2013, pp. 2932-2949.
  • E. Peeters, L. van Oeffelen, M. Nadal, P. Forterre, and D. Charlier, ‘A thermodynamic model of the cooperative interaction between the archaeal transcription factor Ss-LrpB and its tripartite operator DNA’, Gene, vol. S0378-1119(13), Apr. 2013, pp. 00412-5.

The data and results from the first paper can be found here. Those of the second paper can be found here.

A full description of the software is given in "van Oeffelen, L., Peeters, E., Nguyen Le Minh, P., and Charlier, D. (2013). The 'Densitometric Image Analysis Software' and its application to determine stepwise equilibrium constants form Electrophoretic Mobility Shift Assays, PLOS One, vol. 9(1): e85146."

Download and installation

The "Densitometric Image Analysis Software" is made available under the GNU general public license. The last update has been executed on July 2, 2015. You can download the MATLAB source code or, if you do not have MATLAB installed, platform-dependent software. To install the software on

- a (64-bit) Windows platform, first download the corresponding MCR release R2015a from the MathWorks Web site. Then install the Windows version of the Densitometric Image Analysis Software

- a 64-bit Mac: this version needs to be updated and is therefore currently unavailable.

Running the software with demo files

The software requires an EMSA or gel image and an excel file containing the experimental data as inputs. Example files are given in Download and unzip this folder, and run the Densitometric Image Analysis Software. Follow the sequence of analysis steps indicated in the graphical user interface at the left, using data.xlsx as a data file and selecting one of the images, to test if the software is correctly installed. The output of the software consists of estimated stepwise equilibrium constants and standard deviations. This output is shown by the software and is also automatically written to the data file on a sheet named 'Results'.

If you encounter any problems, please do not hesitate to contact the author, dr. ir. Liesbeth van Oeffelen.

Analyzing your own data

To analyze your own data, you should follow the next steps:

1) Scan your image together with a calibrated step tablet with a scanner such as the Microtek Bio-5000 scanner, and crop the image and the step tablet. Make sure that you leave some white space at the upper and lower side of the image, but not at the left or the right, as illustrated by the EMSA in the demo file. Do not leave any white space around the step tablet.

2) Put these pictures in the same folder with your data file. To write this data file, use data.xlsx in as a template and add your own data.

3) Run the Densitometric Image Analysis Software.

Some interesting details:

  • The band boundaries will automatically be saved in the results file as well, so they can be read together with the data file if you want to check your data analysis afterwards. If you want to remove these boundaries, you can remove the corresponding entry in the results file.
  • If you want to draw band boundaries immediately without indicating areas, you can draw the first point at the position where you want to place the vertical line, and the second somewhere at the right of the first one.
  • When indicating areas to determine band boundaries, the program makes sure that the intensity cannot be under zero. Hence, if you select a point under the figure, it will be counted as a zero intensity.
  • If you perform band subtraction, make sure that this band is selected between two vertical lines, i.e., completely within the smear between two bands, or within a band.
  • Analyzed phosphorimages should have gray values proportional to counts.
  • In our work, we used 32P, with 10.000 cpm per lane. In case of much lower exposures in combination with intensifying screens, take into account that autoradiographic films can be less sensitive for low-intensity bands, since Ag atoms may be reconverted to Ag+ ions in between the passage of 2 photons (see this reference). Be aware of this if you observe binding constants significantly increasing with protein concentrations. This issue may be overcome by preflashing the film.

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