In this experiment, image processing techniques are applied to study the western blot analysis.
- To compare the amount of protein in the given sample by analyzing the intensity of the band produced in the gel.
Visualization and image analysis methods are critical for understanding various features of cell biology, molecular biology and neuroscience applications. The recent advances in the biological and medical field have made the image analysis increasingly important in this discipline. A number of freely available online tools are currently available to analyze the biological images. In this section, the densitometry analysis, that is, the determination the variations in the density of protein samples by comparing it with a certain standard value/ determining the intensity of the specific bands in the western blot is done using the software Image J. It is basically a public domain Java image processing program that was developed at the National Institute of Health. The basic steps involved in densitometry analysis of proteins are as follows:
- Blot images are imported into the software and the contrast is adjusted in such a way to obtain a clear western blot image.
- Using rectangular selection tool, area around each bands to be analyzed are selected.
- The bands are then analyzed.
- The intensities of the bands are displayed and the relative concentration of protein in the sample is quantified.
Western blotting is a core technique in molecular biology and cell biology, which is used to detect the presence of specific protein in a complex mixture extracted from a wide variety of biological samples, such as tissue homogenates or cultured cells. It depends on the ability of a particular protein to bind with specific antibodies. The technique is otherwise known as immunoblotting or protein blotting. The proteins are separated on a SDS- polyacrylamide gel (SDS PAGE) on the basis of their molecular size; where the smaller proteins migrate faster in the gel while the larger proteins will move slower in the gel. That is, the larger the protein, the slower it moves on the gel. When the western blotting technique and PAGE is combined together, it will help in the powerful analysis of the protein information such as mass, charge, purity, etc. Following the PAGE, due to the electrophoretic mobility, the proteins are transferred from the electrophoresis gel to a nylon membrane. Due to the applied electric voltage, the proteins move out of the polyacrylamide gel and become tightly attached onto the surface of the membrane. Meanwhile, the blocking buffer (3% skimmed milk solution) helps to "block" the non-specific binding of proteins. The next step is to probe the blocked membrane with primary antibody that recognizes specific proteins or its epitopes. But the primary antibody which recognizes the target protein is not directly detectable. Hence nitrocellulose is then incubated with tagged secondary antibodies, which is specific for the primary antibody. The secondary antibodies are coated with an enzyme that react with a chromogenic substrate, will result in color reaction. This helps in an easy detection and documentation for Western Blotting.
For more details refer Cell biology Lab I, Western Blotting
Densitometry analysis of Western Blot
The quantitative analysis of the protein expression gains more importance with the development of interdisciplinary sciences such as system biology. ImageJ program inspired from NIH (USA) is probably the cheapest and easiest way to quantify western blots. The ImageJ tool helps in the measurement of protein expression levels, intensities of specific bands, and densitometry analysis of corresponding protein of interest in the given sample. The ability to quantify the intensity of Western blot for the statistical analysis makes densitometry an important tool for the researchers. The determination of area under the curve (Peak) helps to a make relative comparisons of band intensity between the different lanes in the gel.
While analyzing a western blot image, it is important to load similar quantities of protein samples in all the wells, so that the comparison of bands becomes more feasible. This will avoid many of the experimental errors and thus helps to analyze the gel in a biological context.