Western blotting is a technique used in both molecular and cell biology. It allows for the identification of specific proteins, within a more complex mixture of the proteins that have been extracted from a cell. There are three specific elements used to accomplish the task, and the information provided from this type of blotting can be crucial to determining the next steps to take in an experiment or in the treatment of a condition or problem. A strong understanding of this technique is required for use. Here is what to consider.
History of the Western Blot
The technique of Western blotting got its name because it was similar to a technique called Northern blotting, which was developed by another scientist a few years prior. The Northern blotting was a take on the Southern blotting technique, that had also been previously developed. Both of those techniques used radio-labeled DNA and gel. By working to create a replica of the gel that was solid-phase, W. Neal Burnette developed the Western blog (also called the immunoblot). It became a popular option over time.
Separation by Size
After the proteins are extracted from the cells, the first step in western blotting is separation of the proteins by size. This allows the proteins to be grouped, in order to better categorize and study them. When looking for a specific protein in the sample, this size grouping also narrows down what is being examined. This can allow for quicker identification of particular protein types, thus making it easier to discover whether the looked-for proteins are present. If found, they will provide important knowledge.
Transfer to a Solid Support
After the proteins are separated by size, they can be transferred to a solid support. This is often done through the use of gel and nitrocellulose paper. With western blotting, solidifying the gel and being able to transfer the collected protein to the paper is one of the key areas to get proper results. Without the properly solidified gel, for example, It is likely that the transfer will be unsuccessful. It is vital not to rush the process, and to allow the gel to work as intended in order to have the strongest possibility of success.
Marking Target Protein
The marking of the target protein requires a primary and secondary antibody. Both are needed in order to properly visualize the protein. However, the data that is created with western blotting is considered to be only semi-quantitative in nature. It does provide a comparison of the levels of protein, but it does not create a measure of quantity that is absolute. The reasons behind this are twofold.
First, there is a variation in the transfer and loading rates between the samples in separate lanes, and that makes them different on the separate blots, as well. Second, the detection signal that is generated by the discovery of the proteins is non-linear when examined across the entire range of samples. This non-linear signal is not able to be used in order to fully model the concentration.
Troubleshooting and Difficulties
The Western blot technique is not without risks and difficulties, but there are many options for troubleshooting the most frequent concerns. Most typically, the problems are with faint or weak bands, no bands, unexpected bands, high background, or patchy spots. This can come from the antigen, buffer, or antibody used, and can also be caused by protease degradation.
Uneven spots are generally a problem created during transfer, and a concentration of the antibody that is too high can cause background issues. The usefulness of this technique generally makes it worth troubleshooting the issue, so the experiment can proceed for the future.