How to Quantify Electrophoresis Gels in Images

Electrophoresis is an essential technique for gene cloning and other advanced molecular biology experiments. Scientists routinely use electrophoresis for one purpose only, to separate DNA, RNA or proteins into their individual molecular components within a gel slab. This procedure allows scientists to determine how much of a molecule is present in a sample and how big these molecules are. To do this, the sample is allowed to migrate down the length of the gel, during which it splits into bands of different sizes at distinct points inside the gel. Finally, the gel containing the migrated molecules is photographed. It is this image that must be quantified for data analysis purposes.

Things You'll Need

• Image analysis software

Instructions

Electrophoresis Gel Image Quantification

1. Choose a suitable image analysis software. This is mandatory, therefore access to densitometry software such as KODAK DirectView EVP Plus and Carestream Molecular Imaging must be obtained beforehand. Alternatively, cheaper software (e.g., Adobe Photoshop) and open-source software tailored specifically to scientific image analysis (e.g., ImageJ) are also available. ImageJ is a free, full-featured package common in molecular biology laboratories and simple to operate. For the purpose of this article, ImageJ will be used as an example. However, the principles in each step are universal and easily applicable to all types of image analysis software.

2. Convert the image into a usable format. ImageJ is compatible with many imaging machines and cameras used in laboratories that carry out electrophoresis. However, it is common practice to create .JPEG, .TIFF or other universal formats of the image to be analyzed.

3. Import the image(s) into the image analysis software. Some image analysis software programs cause the imported images to have lower resolution, different contrast and other image quality problems; therefore, it is a good idea to import all formats and choose the one that has the highest quality in the chosen software. ImageJ does not have the resolution problem and preserves the quality of the raw data.

4. Convert the image color to \"grayscale.\" In ImageJ, select the \"Image\" tab and in the menu, click \"grayscale.\"

5. Set the correct measurement parameters for the analysis. On the Analyze tab, select \"Set Measurements,\" then check the boxes for \"Area,\" \"Mean Gray Value\" and \"Integrated Density\" in the drop-down menu.

6. Set the correct scale for the analysis. In the example software, click \"Analyze,\" then \"Set Scale,\" and in the space for Unit of length, type in \"pixels.\"

7. Invert the colors for visual clarity. Under the Edit tab, select \"Invert\" so that the light areas become dark and vice versa.

8. Select the desired bands in the image. On the tool palette, select the \"Freehand Selection\" tool and drag the mouse pointer along the border of either the first band or the band that is the control experiment. Select only the band, and avoid all background shading, which is essentially noise.

9. Measure (that is, quantify) the selected area. Press \"m,\" and in the Results window that appears find the measurements for Integrated Density, Area and Mean Gray Value, etc. Repeat the above steps until all bands have been measured. Then copy all the values to a spreadsheet. The quantified images are now ready for data analysis in the spreadsheet program.

References

• "The Protein Protocols Handbook?"; John M. Walker; 2002
• "Electrophoresis in Practice"; Reiner Westermeier, Sonja Gronau; 2005
• Quantifying Western Blots without Expensive Commercial Quantification Software

Resources

• Read this Article in Spanish
• DNA Quantification by Gel Densitometry with Norgen DNA Ladders