Journal of Comprehensible Results

Western blots can be used to determine the levels of a specific protein. The brains of the rat pups were subjected to a western blot after being homogenized to create a uniform solution. A western blot is composed of four main steps: separation, transfer, staining, and visualization as shown below[11].

First, the solution was separated through SDS PAGE: SDS causes the proteins in solution to lose their shape and have a negative charge, so that when they are placed in a gel all proteins will travel towards the positive end. Proteins that are larger will move more slowly, so the end result are proteins separated by molecular weight. Because it is difficult to interact with proteins while they are in a gel, they are transferred to a membrane.

Next, two antibodies are introduced to target the protein of interest; for example, to target myelin associated glycoproteins (MAG) an anti-MAG antibody would be used. The first antibody introduced is called the primary antibody and is grown to target and attach to a particular protein. After the primary antibody binds to the protein of interest, there is still no way to visualize the results so a secondary antibody that targets the primary antibody is introduced. In this experiment, the secondary antibody contained horseradish peroxidase which can react with its substrate, SuperSignal West Dura reagent, to create chemiluminescence which is then visualized on film in a dark room and then the intensity of the bands that are produced is read by an imaging program.

From the program, results similar to what is shown on the right will be outputted. Results are given as a percent of the control, meaning that changes in protein level can be measured but the actual number or amount of a particular protein is not being measured. In this example, the treatment group given 0.3 mg/kg/day of buprenorphine did not differ significantly from the control and the group that received 1 mg/kg/day of buprenorphine showed a significant decrease in myelin associated glycoproteins at 12 days of age.

Signficance is determined by a statistical test. Even if there appears to be a difference between the control and a treatment group, they are only considered truly different if it can be proven statistically. In the example on the left, significance is indicated by the ** above the bar. In all other graphs, significance is indicated by a red star above the bar.

Example Western Blot Results Chemiluminescent bands are created be the secondary antibody and then an imaging program reads the intesity of those bands. A darker band indicates a higher level of the protein of interest. These intensities are then put in terms of the control.If the band produced is darker than the control, than the protein level increased; whereas, if the band is lighter than the protein level decreased.