Western Blotting: Molecular biology on Hight

Some clinical research and medical therapies arising from molecular biology are covered under gene therapy whereas the use of molecular biology or molecular cell biology in medicine is now referred to as molecular medicine.

Molecular biology also plays important role in understanding formations, actions, and regulations of various parts of cells which can be used to efficiently target new drugs, diagnose disease, and understand the physiology of the cell.

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Macromolecule blotting and probing

Northern blotting

The northern blot is used to study the expression patterns of a specific type of RNA molecule as relative comparison among a set of different samples of RNA. It is essentially a combination of denaturing RNA gel electrophoresis, and a blot. In this process RNA is separated based on size and is then transferred to a membrane that is then probed with a labeled complement of a sequence of interest.

The results may be visualized through a variety of ways depending on the label used; however, most result in the revelation of bands representing the sizes of the RNA detected in sample. The intensity of these bands is related to the amount of the target RNA in the samples analyzed.

The procedure is commonly used to study when and how much gene expression is occurring by measuring how much of that RNA is present in different samples. It is one of the most basic tools for determining at what time, and under what conditions, certain genes are expressed in living tissues.

Western blotting

In western blotting, proteins are first separated by size, in a thin gel sandwiched between two glass plates in a technique known as SDS-PAGE. The proteins in the gel are then transferred to a polyvinylidene fluoride (PVDF), nitrocellulose, nylon, or other support membrane.

This membrane can then be probed with solutions of antibodies. Antibodies that specifically bind to the protein of interest can then be visualized by a variety of techniques, including colored products, chemiluminescence, or autoradiography. Often, the antibodies are labeled with enzymes. When a chemiluminescent substrate is exposed to the enzyme it allows detection.

Using western blotting techniques allows not only detection but also quantitative analysis. Analogous methods to western blotting can be used to directly stain specific proteins in live cells or tissue sections.

Eastern blotting

The eastern blotting technique is used to detect post-translational modification of proteins. Proteins blotted on to the PVDF or nitrocellulose membrane are probed for modifications using specific substrates.

Southern blotting

Named after its inventor, biologist Edwin Southern, the Southern blot is a method for probing for the presence of a specific DNA sequence within a DNA sample. DNA samples before or after restriction enzyme (restriction endonuclease) digestion are separated by gel electrophoresis and then transferred to a membrane by blotting via capillary action. The membrane is then exposed to a labeled DNA probe that has a complement base sequence to the sequence on the DNA of interest.

Southern blotting is less commonly used in laboratory science due to the capacity of other techniques, such as PCR, to detect specific DNA sequences from DNA samples. These blots are still used for some applications, however, such as measuring transgene copy number in transgenic mice or in the engineering of gene knockout embryonic stem cell lines.

Automation in Western Blot Technique

Western blot is the major analytical tool to assess the molecular function in basic cell biology research, drug discovery and pathology. There have been significant improvement in the technical aspect of western blot which has led to improvement in efficiency, reproducibility, ease of use. For example, iBind Western Systems launched by ThermoFisher Scientific Corporation which is and automated western blot device which does not require continuous monitoring. Further the turnaround time is reduced compared to traditional method where it involves the various steps and four hours for final results. In iBind all the solution is required to be loaded rest all process is automated by sequential lateral flow technology (SLF) which does not require any electricity or batteries. The significant advantage provided by automated western blot system will contribute in the growth of the western blot market.