In the field of molecular biology, different blotting techniques are used to detect, identify and quantify the presence of a specific protein or to identify nucleic acid sequences.
Blotting refers to the process during which macromolecules are transferred from a gel onto the solid surface of a membrane, which will in other words, aspirate the information from the surface of the gel. Three main techniques are used: Southern, Northern and Western Blot.
All three of them are based on similar protocols, but the outcome varies, as well as some of the steps of the protocol. In this article we will get to know the goals and steps of each technique.
Southern Blotting was first discovered and set up by Edward M. Southern in 1975. It is a procedure which has been developed to identify particular sequences of DNA.
The protocol begins with the extraction of DNA from cells, which then leads to the production of DNA fragments. These fragments are then placed on the top of an agarose gel. This type of gel is particularly used for nucleic acid separation because of its reticulation ability, which means that the gel is able to easily separate bigger and smaller molecules.
Because they are charged negatively, the nucleic acids will respond to the electric field created by the electrophoresis. Based on their sizes, smaller molecules will migrate further down the agarose gel sheet and on the contrary, the bigger ones will remain on the upper part of the gel.
After the separation is complete, the DNA on the gel is transferred onto a nylon membrane. This membrane is then incubated in a solution which contains a DNA probe, which is complementary to the target DNA. In order to easily detect and reveal the targeted molecules, the probes can be fluorescently labelled and visualised thanks to X-Ray film, or they can also be enzymes able to generate a chemiluminescent signal which will reveal the target DNA.
Southern blotting protocol is used for instance to test paternity, to identify victims or criminals through blood or other samples containing DNA. It can also be used to detect DNA mutations, deletions or gene rearrangements, as well as to detect an infection or a prenatal genetic disease.
Northern Blotting is a procedure which is applied to detect, and identify specific RNA sequences. First, the protocol requires RNA isolation. Depending on the size of the sequence, the separation phase will vary.
Large sequences are separated by electrophoresis on formaldehyde agarose gel for example. This gel is supposed to let the RNA sequence the way it has been isolated and to prevent normal base pairing of nucleic acids.
Smaller sequences are applied on a polyacrylamide gel. Electrophoresis reaction and the separation process can now begin. Afterwards, the RNA is transferred from the gel onto a nylon membrane. The membrane is then incubated with a radioactively labelled RNA probe.
Which allows the detection of the targeted elements, visually identified thanks to chemiluminescence provided by radioactively labelled RNA probes.
This technique is mainly used in the field of gene expression studies. Our DNA makes us who we are. It delivers instructions to our cells in order to produce everything they need so that our body functions properly. When this information stored in our DNA is converted into instructions to make proteins for example, this is what gene expression is.
Western Blotting is a procedure meant to identify the presence, the size and abundance of specific proteins in a sample. The technique is usually used on small sequences.
First, the protocol consists in preparing the sample by isolating the proteins from the cells. To do so, a lysate preparation meant to separate the proteins from the other cell components is applied.
Once the proteins are isolated correctly, each of them needs to be denatured by a reducing agent, called SDS. When subjected to heat, SDS is going to denature the protein and give it a negative electrical charge, which will later allow the migration of the gel. This denaturing process is going to unfold the proteins and determine their molecular weight.
This step is crucial for the following process. Contrarily to DNA and RNA, protein conformation can have an impact on the migration of the proteins in the polyacrylamide gel, therefore, they need to be denatured. Once the protein samples are ready, the separation phase can start with electrophoresis. Being quite similar to agarose gel, polyacrylamide gel is used here because it provides more accurate results, as it is chemically more stable than agarose.
All the protein samples are deposited on wells at the top of the gel. Next to these samples is added a reference sample of several coloured proteins whose molecular weights are known, in order to compare and estimate the weight of the proteins contained in the other samples.
Once the proteins have been denatured by SDS, they end up being negatively charged. When the electrical current is activated, the smaller molecules will go further down the gel whereas the bigger ones will remain at the top, while the proteins from the reference sample will separate according to their molecular weights.
After all the molecules are separated, they are transferred to a nitrocellulose membrane in order to fixate the proteins and help with the following process. The membrane is incubated with a blocking agent, reducing the risk of binding undesired antibodies to proteins.
The membrane is incubated with a primary antibody which only binds to specific proteins; a few moments later, the membrane is incubated with a secondary antibody. Antibodies are either chemiluminescent or fluorescent or affected with a colour, allowing biologists to clearly identify the presence or absence of targeted proteins.
Western blotting technique is often used to determine the presence of certain proteins. The procedure can be used to diagnose HIV infection for instance. Or to detect defective proteins, Hepatitis B, Creutzfeldt-Jakob disease, Lyme disease and Herpes infection.
Southern, Northern and Western blot are three identification techniques which are quite similar in the making process. The three techniques all use the same type of procedure: starting with the extraction of a sample, continuing with the electrophoresis separation phase on a gel, then transferring of the sample to a membrane and the detection of the desired molecule.
They are all meant to identify a specific macromolecule within a single sample and they all apply to biotechnology and medicine.
Distinguishing the three techniques might not be so easy. However, the main difference that has to be noted is the molecule that each technique is meant to identify. Southern blot detects specific DNA sequences, Northern blot detects particular RNA sequences, and Western blot detects specific proteins. All these techniques are complementary.
Furthermore, it is worth saying that, even though the methods used are very similar, the processes all differ a bit from the others. They will not use the same gel for instance, or the same type of membrane to conduct the detection.