Blotting 101: Southern Blot vs Western Blot

If you’re studying biology or biotechnology, you’ve probably come across terms like Southern blot and Western blot in your textbooks or lectures. While the names may sound similar, the process of each is different and they detect different targets. The Southern blot technique, developed by Edwin Southern in the 1970s, is primarily used for the identification of specific DNA sequences within a complex mixture, allowing researchers to visualize the presence of particular genes or genetic variations. On the other hand, the Western blot technique focuses on proteins, utilizing protein gel electrophoresis to separate them based on their size before transferring them onto a membrane for detection. This method is crucial for studying protein expression, modifications, and interactions in various biological contexts. Understanding the distinct purposes and methodologies of these techniques can significantly enhance your research and comprehension of molecular biology.

To reiterate, the main difference between Southern blotting and Western blotting is the type of target. A Southern plot helps scientists find a specific DNA sequence in a sample. This is helpful if you are trying to figure out whether a certain gene is present in someone’s DNA and has real life applications such as checking for genetic mutations, DNA fingerprinting, and studying gene structure.

The steps of a Southern blot are as follows

1. Extract the DNA from your sample.
2. Digest the DNA. This is a process of cutting the DNA into smaller pieces using restriction enzymes.
3. Separate the DNA fragments using agarose gel electrophoresis (this sorts them by size).
4. Transfer the DNA from the gel onto a special membrane (like sticky paper). Southern blotting typically uses nylon or nitrocellulose membranes. This process typically uses an electrical current or capillary action to transfer the DNA.
5. Hybridize the membrane is incubated with labeled DNA probes (a small piece of DNA that binds only to your target sequence). Probes have a very specific sequence of nucleotides, which is chosen by the experimenter, as well as an attached fluorescent or radioactive molecule that serves as a label.
6. Visualize the result by highlighting probe labeled DNA bands. This can either radioactive, fluorescent, or enzyme-based, however most Southern blotting probes are radioactively labeled which are visualized using x-ray film.

Western blots on the other hand detect specific proteins. This technique can show whether a protein is present, how much of it is there, and sometimes whether it’s been modified. This is useful for a multitude of reasons such as diagnosing diseases, checking for protein expression, and identifying protein changes which could indicate certain conditions such as cancer.

The process of performing a Western blot is:

1. Extract proteins from your sample (e.g., cells or tissues).
2. Separate them by size using SDS-PAGE (polyacrylamide gel electrophoresis)
3. Transfer the proteins to a membrane using either capillary action or an electrical current. Membranes typically used in Western blot are either nitrocellulose or PVDF.
4. Block the membrane so that antibodies don’t stick randomly. This involves placing the membrane in a blocking buffer containing detergents and blocking proteins that will bind to all unoccupied sites on the membrane.
5. Add a primary antibody that binds specifically to the protein you want to detect.
6. Add a secondary antibody that recognizes and binds to the primary antibody and has a label that is used for detection.
7. Visualize the protein using a detection system such as chemiluminescence or a noticeable color change

Both types of blot are useful lab techniques that are used in diagnostic facilities and labs across the globe. Try it for yourself and check out Edvotek kit 207 for a Southern blot and Edvotek kit 317 for a Western blot!