ABSTRACT:
In this article, we will discuss about the blotting techniques, their principles and fabulous procedures. Blotting techniques play a pivotal role in molecular biology, enabling scientists to analyze and visualize biomolecules with precision. These techniques have revolutionized the way we study DNA, RNA, and proteins, offering valuable insights into cellular processes. In this article, we will explore the fundamentals of blotting techniques, their applications, and advancements in the field. We will also provide references to learn and acknowledge the importance of blotting techniques.
INTRODUCTION-BLOTTING TECHNIQUES:
The word blot mean dark mark , stain or ink , thus blotting a technique of transferring the
sample onto the membrane in order to analyze or detect. It has following types: Southern blotting, Western blotting and Northern blotting. The technique was given in 1970s and it proved to be very serviceable in the biological fields. Recent years have witnessed advancements in blotting techniques, with the integration of innovative technologies. For instance, quantitative PCR (qPCR) and RNA-Seq have complemented traditional Northern blotting, offering higher sensitivity and specificity in RNA analysis. Similarly, advancements in mass spectrometry have expanded the capabilities of protein analysis, providing a more comprehensive understanding of the proteome.
TYPES OF BLOTTING TECHNIQUES:
1. SOUTHERN BLOTTING:
It is defined as;
“Southern blot is a laboratory technique used in molecular biology to detect and analyze specific
DNA sequences.” It named after the scientist Edwin Southern, who introduced this method in the 1970s. This technique helps to analyze and characterize DNA by separating it and then transferring it onto a solid support, which can be a nitrocellulose or nylon membrane.
PRINCIPLE:
Southern blotting is based on the principle of analyzing specific DNA sequences fragment in a
sample. Firstly, DNA extracted from cells or tissue samples using various methods, such as
enzymatic digestion or chemical lysis. Then DNA of genome is first fragmented using restriction
enzymes and then separated through gel electrophoresis. The denatured DNA fragments are
transferred from the gel to a membrane, where a labeled DNA probe, which is complementary to
the target sequence, hybridizes with specific fragments. These probes can be radioactive or
fluorescently labeled (produce specific light). Detection of the labeled probes allows to identify
and analyze specific DNA fragments on the membrane.
PROCEDURE:
The first step in Southern blotting is to extract the DNA from the samples of interest. This could
involve isolating genomic DNA from cells, tissues, or even viruses using different extraction
techniques. The extracted DNA then cut into smaller pieces by using restriction enzymes. These enzymes recognize specific DNA sequences and cleave the DNA at those sites, generating fragments of varying lengths. The DNA fragments loaded onto an agarose gel and electrophoresis done. In this step, an electric field applied, due to which negatively charged DNA molecules start to move
through the gel matrix towards positive end. Smaller fragments move faster and travel than
larger fragments which move very slowly.
Transferring onto Membrane, the water move from tray upto tissues via capillary action .When water has travelled half of the pile remove the membrane . The DNA has also travelled along the capillary action. The membrane has now DNA that may not be visible. The membrane with transferred DNA fixed on place to prevent loss of DNA. Mostly UV light used. The membrane exposed to UV light which form covalent bonding between the DNA and the membrane. Membrane baked at a high temperature up to 80-120°C. This process helps the DNA to adhere to the membrane.
The transferred DNA fragments on the membrane is treated with labeled probes that are complementary to the target DNA sequences. These probes hybridize with the specific DNA fragments of interest and form stable double-stranded complexes with DNA fragments. After hybridization the membrane goes through washing steps to remove unbound or non-specifically bound probe molecules. Then detection and result analysis take place.
2. NORTHERN BLOTTING:
It is a blotting technique that is developed by Ames Alvin, David camp and George at Stanford
University in 1979. It a technique that used to detect the RNA or it can be performed to determine the gene expression. It is very similar to the southern blotting.
PRINCIPLE:
We isolate the sample i.e. RNA from the cell and denature it in order to break the secondary
structures. Run it on the gel by electrophoresis. Once we get the sample on gel transfer it on the
nitrocellulose membrane. Wash it after hybridization to remove unspecific binding. Detect the
results by autoradiography or by checking fluorescence.
PROCEDURE:
The isolation of RNA is the first step in this technique. In order to denature RNA and break the hair pin loop like structures by various enzymes. We can denature it with heat treatment at 70 degree for 5 mint. RNA loaded on nitrocellulose gel. We put the sample and it will run but the band may or may not appear , smaller fragment travel to cover long distance. After gel electrophoresis, the RNA fragments transferred from the gel onto a solid support which is typically a nitrocellulose. This transfer can achieved by capillary action or by applying an electric field during electro blotting. The UV light used to fix RNA on the membrane. The membrane blocked by bovine serum albumin or dry milk to prevent any nonspecific binding. The probe attached onto the membrane. Then washing and detection occur.
3. WESTERN BLOTTING:
“Western blotting, which is also known as protein immunoblotting, is a laboratory technique
used to detect specific proteins in a given sample.” This technique basically combines the principles of gel electrophoresis and immunoblotting to separate and identify proteins on the basis of their molecular weight and antigenic properties. By using antibodies that bind to specific proteins, it is possible to study protein expression, post-translational modifications and protein-protein interactions, and post-translational modifications.
PRINCIPLE:
The principle of Western blotting based on the separation of proteins by gel electrophoresis and
then transfer to a membrane and detection is done by using antibodies. Specific proteins are
probed with complementary antibodies which leads to the formation of detectable signals. It
allows the analysis of protein expression, their size and mutations in given samples.
PROCEDURE:
Process is start by extracting protein from cells or tissues. Extraction done by using suitable
lysis buffer to release the proteins of interest. Next step is to prepare a polyacrylamide gel and load the protein samples into wells. An electric current applied to separate the proteins based on their size. After the separation, the proteins separated from the gel onto a solid support membrane, such as a nitrocellulose or PVDF membrane. Then membrane incubated by using blocking solution (often 5% milk or BSA) to prevent binding of non-specific antibodies. Incubate the membrane with primary antibodies which is specific to the target proteins. After washing, the membrane incubated conjugated to an enzyme (e.g., HRP or alkaline phosphatase) which is complementary to primary antibody. Target proteins identified by using proper detection methods. Apply a substrate that reacts with the enzyme on the secondary antibody. Then analysis take place.
CONCLUSION-BLOTTING TECHNIQUES:
Blotting techniques have been instrumental in unraveling the intricacies of molecular biology, enabling researchers to decode the language of nucleic acids and proteins. From the classic Southern blotting to the cutting-edge applications in genomics and proteomics, these techniques continue to shape the landscape of biological research. As technology evolves, so too will the potential for deeper insights into the molecular mysteries that govern life.
REFERENCES:
Alwine, J. C., Kemp, D. J., & Stark, G. R. (1977). Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proceedings of the National Academy of Sciences, 74(12), 5350–5354. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC431715/
Towbin, H., Staehelin, T., & Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences, 76(9), 4350–4354. https://pubmed.ncbi.nlm.nih.gov/388439/
Southern, E. M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. Journal of Molecular Biology, 98(3), 503–517. https://pubmed.ncbi.nlm.nih.gov/1195397/
Bustin, S. A. (2000). Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. Journal of Molecular Endocrinology, 25(2), 169–193. https://pubmed.ncbi.nlm.nih.gov/11013345/
