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Blotting

This document summarizes different blotting techniques used to identify proteins and nucleic acids, including Southern blotting, Northern blotting, and Western blotting. Southern blotting is used to analyze DNA fragments and involves transferring DNA from a gel to a membrane and using a labeled probe. Northern blotting analyzes gene expression by separating RNA, transferring it to a membrane, and using a probe. Western blotting identifies specific proteins by separating proteins via electrophoresis, transferring them to a membrane, and using antibodies to detect the target protein. These techniques are important tools in molecular biology and clinical research.

Education◦
Blotting: Southern, Northern and Western Blotting Mrs. Praveen Garg VITS College, Satna
INTRODUCTION • Blotting techniques are used to identify proteins and nucleic acid sequences. • They have been developed to be highly specific and sensitive technique. • It become important tools in both molecular biology and clinical research. • This process can be done after the gel electrophoresis, by transferring the molecules from the gel onto the surface of blotting membrane or solid support. • These transferring molecules visualized by using different stain Ethidium bromide, Crystal violet, Safranine and Ossmium tetroxide etc
TYPES OF BLOTTING There are basically 4 types of blotting: • Southern blotting • Western blotting • Northern blotting • Eastern blotting
SOUTHERN BLOTTING • Southern blotting is named after Edward M. Southern in 1975. • This method is used for analysis of DNA fragments in a complex mixture. • Nitrocellulose membrane used in this method for transferring the band. • In this method, DNA-DNA hybridization take place. • It is also called as southern hybridization
METHODOLOGY • Firstly, large weighted DNA is cut into small fragments by using Restriction endonucleases enzyme. • Then, these fragments are electrophoresed on separating gel so that they can separate according to their size. • If DNA fragments are much larger in size so firstly the gel should be treated with HCl, causes depurination of DNA fragments. • The separated DNA molecule are denatured by alkali treatment and placed a nitrocellulose membrane over the separating gel. • All DNA fragments present on gel is transferred to the membrane.
• After that the membrane is exposed to ultraviolet radiation so that the fragments are permanently attached to the membrane. • Then the membrane is exposed to hybridization probe. But the DNA probe is labeled so that it can easily detect, when the molecule is tagged with a chromogenic dye. • After hybridization process, excess probe is washed away by using buffer and it can be visualized on X-ray film with the help of autoradiography.
APPLICATIONS • It is used in the technique called RFLP (Restriction fragment length polymorphism) mapping. • Also used in phylogenetic analysis. • To identify the gene rearrangements . • It is important for gene cloning.
• The northern blot technique was developed in 1977 by James Alwine. • The northern blot or RNA blot, is a technique used in molecular biology to study gene expression by detection of RNA (or isolated mRNA) in a sample. • Northern blotting involves the use of electrophoresis to separate RNA samples by size. • Its detection can be done with a hybridization probe complementary to part of or the entire target sequence. • The term 'northern blot' actually refers to the capillary transfer of RNA from the electrophoresis gel to the blotting membrane. NORTHERN BLOTTING
• Blotting procedure starts with extraction of total RNA from a homogenized tissue sample or from cells. • Eukaryotic mRNA can then be isolated through the use of oligo (dT) cellulose chromatography to isolate only those RNAs with a poly(A) tail. • RNA samples are then separated by gel electrophoresis. • Since the gels are fragile and the probes are unable to enter the matrix, the RNA samples, now separated by size, are transferred to a nylon membrane through a capillary or vacuum blotting system. METHODOLOGY
• A nylon membrane with a positive charge is the most effective for use in northern blotting since the negatively charged nucleic acids have a high affinity for them. • The transfer buffer used for the blotting usually contains formamide. • Once the RNA has been transferred to the membrane, it is fixed to the membrane by UV light or heat. • After a labeled probe has been hybridized to the RNA on the membrane. • The membrane is washed to ensure that the probe has bound specifically. • The hybrids are then detected by X-ray film and can be quantified by densitometry.
• Northern blotting allows to observe a particular gene's expression pattern between tissues, organs, developmental stages, pathogen infection. • The technique has been used to show over expression of oncogenes and downregulation of tumor-suppressor genes in cancerous cells when compared to 'normal' tissue. • The variance in size of a gene product can also indicate deletions or errors in transcript processing. • By altering the probe target used along the known sequence it is possible to determine which region of the RNA is missing. APPLICATIONS
• The western blot or western blotting, is a widely used analytical technique in molecular biology and immunogenetics. • It detect specific proteins in a sample of tissue homogenate or extract. • The sample undergoes protein denaturation, followed by gel electrophoresis. • Western blotting is also called protein immunoblotting because an antibody is used to specifically detect its antigen. WESTERN BLOTTING
The technique consists of three major processes: • Separation of proteins by size (Electrophoresis). • Transfer to a solid support (Blotting) • Marking target protein using a proper primary and secondary antibody to visualize (Detection).
• Electrophoresis used to separate proteins according to their electrophoretic mobility which depends on charge, size of protein molecule and structure of the proteins. • Proteins are moved from within the gel onto a membrane made of Nitrocellulose (NC) or Polyvinylidene difluoride (PVDF). • Proteins combine with nitrocellulose membrane based on hydrophobic interaction (Blotting). • For detection of the proteins primary antibody and enzyme conjugated secondary antibody are used. • On addition of substrate, a substrate reacts with the enzyme that is bound to the secondary antibody to generate colored substance, namely, visible protein bands.
• In this technique a mixture of proteins is separated based on molecular weight, through gel electrophoresis. • These gel are then transferred to a membrane producing a band for each protein. • The membrane is then incubated with labels antibodies specific to the protein of interest. • The unbound antibody is washed off. • The bound antibodies are then detected by developing the film. • As the antibodies only bind to the protein of interest, only one band should be visible. • The thickness of the band corresponds to the amount of protein present.
• It utilizes SDS-PAGE (Sodium dodecyl sulphate polyacrylamide gel electrophoresis) gel electrophoresis. • It can use nitrocellulose membrane and nylon membrane. • After hybridization, antigen-antibody complex is formed. • If the protein is bound by a radioactive antibody, its position on the blot can be determined by exposing the membrane to a sheet of X-ray film, a procedure called autoradiography.
• Identification of a specific protein in a complex mixture of proteins. • In this method, known antigens of well-defined molecular weight are separated by SDS-PAGE and blotted onto nitrocellulose. • The separated bands of known antigens are then probed with the sample suspected of containing antibody specific for one or more of these antigens. • Reaction of an antibody with a band is detected by using either radiolabeled or enzyme-linked secondary antibody that is specific for the species of the antibodies in the test sample. • Estimation of the size of the protein as well as the amount of protein present in the mixture. APPLICATION
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