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Blotting (Southern, Northern and Eastern)

Blotting techniques such as Southern blotting, Northern blotting, and Western blotting allow for the transfer and detection of DNA, RNA, and proteins, respectively. Southern blotting involves separating DNA fragments via gel electrophoresis, transferring them to a membrane, and using a probe for hybridization to detect specific DNA sequences. Northern blotting detects RNA transcripts by separating RNA on a gel, transferring it to a membrane, and labeling it with a probe. Western blotting identifies proteins by separating them on an SDS-PAGE gel, transferring them to a membrane, and using primary and secondary antibodies to detect specific proteins based on antigen-antibody binding. These techniques have various applications in research, diagnostics, and forensics

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BLOTTING TECHNIQUES
What is blotting? Technique for transferring • DNA • RNA • Proteins onto a carrier so they can be separated, and often follows the use of a gel electrophoresis.
TYPES OF BLOTTING TECHNIQUES BLOTTING TECHNIQUES Southern Blot It is used to detect the DNA. Northern blot It is used to detect the RNA. Western blot It is used to detect proteins.
Blotting sheet • Whatman 3mm paper….. world’s most widely used blotting paper.  REASON???? • high quality • purity • consistency
Creating the Sandwich • The sandwich consists of : filter paper Nitrocellulose membrane gel matrix another piece of filter paper
SOUTHERN BLOTTING History: • Named after Sir Edwin Southern • Developed in 1975
SOUTHERN BLOTTING “Used to detect the DNA” • This method Involves:  Separation  Transfer  Hybridization. • This DNA can be: • Single gene • Part of a larger piece of DNA……..viral genome “The key to this method is Hybridization”
Hybridization “Process of forming a dsDNA molecule between a ssDNA probe and a ss-target patient DNA”
PRINCIPLE  The mixture of molecules is separated.  Immobilized on a matrix.  Probe addition to the matrix to bind to the molecules.  Unbound probes are removed. “The place where the probe is connected corresponds to the location of the immobilized target molecule.”
Steps in southern blotting The DNA is digested Fragments Gel electrophoresis Transfer to membrane Probing Autoradiogram
APPLICATIONS Southern blotting is used in: • Gene discovery • Mapping • Evolution • Development studies • Diagnostics • Forensics
Northern Blotting History: Northern blotting was developed by James Alwine and George Stark at Stanford University. Northern blotting is a technique for detection of specific RNA sequences
Steps involved in N.B RNA isolation Loading of sample on Agarose gel Blotting on nitrocellulose membrane Labeling with probe Washing to remove unbound probe Detection by autoradiogram
APPLICATIONS • A standard for the direct study of gene expression at the level of mRNA (mRNA transcripts) • Detection of mRNA transcript size • Study RNA degradation • Study RNA splicing - can detect alternatively spliced transcripts • Study RNA half-life • Study IRES (internal ribosomal entry site) – to remove possibility of RNA digestion vs. 2nd cistron translation.
Western blotting Discovery??? • Dr. Douglas Lake of the University of Arizona School of Medicine's Department of Microbiology and Immunology “A technique in which proteins are separated by gel electrophoresis and transferred to a membrane sheet. A specific protein is then identified through its reaction with a labeled antibody.”
Principle This technique works on the principle on “Antigen-Antibody” relationship
Prerequisite for W.B The SDS PAGE technique is a prerequisite for Western blotting. “SDS (sodium dodecyl sulfate) is a detergent (soap) that can dissolve hydrophobic molecules but also has a negative charge (sulfate) attached to it.”
Steps in W.B 1. Gel electrophoresis: The proteins are separated according to size. 2. Membrane Transfer: Transferring to nitrocellulose by applying current. 3. Blocking: Done to prevent non-specific protein interactions between the membrane and the antibody protein.
Blocking in W.B • The blot is incubated with a generic protein (such as milk proteins) which binds to any remaining sticky places on the nitrocellulose. • An antibody that is specific for the protein of interest(the primary antibody - Ab1) is added to the nitrocellulose sheet and reacts with the antigen. • Only the band containing the protein of interest binds the antibody, forming a layer of antibody molecules .
Cont.. • Following several rinses for removal of non- specifically bound Ab1, the Ab1-antigen complex on the nitrocellulose sheet is incubated with a second antibody (Ab2), which recognizes the primary antibody and binds it. • Ab2 is radioactively labeled, or is covalently linked to a reporter enzyme, which allows to visualize the protein-Ab1-Ab2 complex
Applications • To identify the specific proteins • To identify their masses • The confirmatory HIV test to detect anti-HIV antibody in a human serum sample. • The definitive test for Bovine spongiform encephalopathy (BSE, commonly referred to as 'mad cow disease'). • Some forms of Lyme disease testing also employ Western blotting.
Protein gel (SDS-PAGE) that has been stained with Coomassie Blue.
Faster Way to Western Blot • The Quick Western Kit provides a universal detection reagent that can be combined with the primary antibody incubation step, eliminating the need for a secondary antibody incubation step. • This kit works with a variety of primary antibodies. • This reduces the overall time to complete a Western blot.
Standard Western Blot
Blotting (Southern, Northern and Eastern)