In this experiment, Image processing techniques are applied to compare the density of bands on an polyacrylamide gel
To compare the density of bands on an polyacrylamide gel using online software.
Electrophoresis is a technique used to separate and purify macromolecules such as proteins and nucleic acids on the basis of the differences in their size, charge or conformation. The basic principle of all electrophoretic procedure is that the charged molecules in aqueous solution will migrate in an electric field. The rate of migration of samples through the electrical field depends on the net charge of the protein/nucleic acid, the size and shape of the molecules, and also on the ionic strength, viscosity, and temperature of the medium through which the molecules are moving. SDS Polyacrylamide Gel Electrophoresis (PAGE) is a common type of electrophoretic technique useful for estimating the molecular weight of proteins. In SDS-PAGE, the protein mixture is denatured by heating at 100°C in the presence of excess SDS and a reducing reagent (β-mercaptoethanol) is employed to break disulfide bonds. Under these conditions, all reduced polypeptide bind SDS and the SDS-Protein complex forms a rod with negatively charge and similar charged density. The length of the SDS-Protein complex is proportional to the molecular weight of the protein and is separated on the basis of their size. SDS PAGE are mainly used for the estimation of protein size and protein purity, determination of protein subunits, monitoring protein integrity, Comparison of the polypeptide and different samples and Post-electrophoresis applications, such as Western blotting.
For more details refer Molecular Biology Lab II Polyacrylamide Gel Electrophoresis
ImageJ is a java based open source software used for image processing and analyzing of the pictures. It was developed by Wayne Rasband at the National Institutes for Health (NIH, USA). It provides a set of ready-made tools for viewing and interactive manipulation of images. It can read many image formats including TIFF, GIF, JPEG, BMP, DICOM, FITS and ‘raw’. This program is largely platform independent and is easily run on all computer platforms including Windows, Mac, Linux, Unix, and even the Sharp Zaurus PDA. It can display, edit, analyze, process, save, and print 8-bit, 16 bit, and 32 bit images. It can calculate area pixel value statistics of user defined selections. It can measure the distances and angles. It can create density histogram and line profile plots. It supports standard image processing functions such as a contrast manipulation sharpening smoothing edge detection and median filtering.
ImageJ can be used to compare the density of bands obtained from an agarose gel. The results obtained from AGE, PAGE can be easily assessed visually by making comparisons between bands in different lanes which is easily analyzed by ImageJ. This experiment provides a brief tutorial on how to effectively quantify the results obtained from PAGE, AGE etc.