blotting (southern, northern and eastern)
TRANSCRIPT
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BLOTTING
TECHNIQUES
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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.
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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.
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Blotting sheet• Whatman 3mm paper….. world’s most widely used
blotting paper.
REASON????• high quality• purity • consistency
(0.34 mm) is used extensively in electrophoresis for lifting of sequencing gels.
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Creating the Sandwich
• The sandwich consists of :filter paper
Nitrocellulose membrane
gel matrix
another piece of filter paper
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SOUTHERN BLOTTING
History:• Named after Sir Edwin
Southern
• Developed in 1975
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SOUTHERN BLOTTING
“Used to detect the DNA”• This method Involves:SeparationTransferHybridization.• This DNA can be:• Single gene• Part of a larger piece of DNA……..viral genome
“The key to this method is Hybridization”
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Hybridization
“Process of forming a dsDNA molecule between a ssDNA probe and a ss-target
patient DNA”
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PRINCIPLEThe 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.”
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Steps in southern blottingThe DNA is digested
Fragments
Gel electrophoresis
Transfer to membrane
Probing
Autoradiogram
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APPLICATIONS
Southern blotting is used in:• Gene discovery • Mapping• Evolution • Development studies• Diagnostics • Forensics
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Northern BlottingHistory:
Northern blotting was developed by James Alwine and George Stark at Stanford University.
Northern blotting is a technique for detection of specific RNA sequences
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Steps involved in N.BRNA isolation
Loading of sample on Agarose gel
Blotting on nitrocellulose membrane
Labeling with probe
Washing to remove unbound probe
Detection by autoradiogram
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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.
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Western blottingDiscovery???• 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.”
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Principle
This technique works on the principle on
“Antigen-Antibody” relationship
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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.”
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Steps in W.B1. 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.
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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 .
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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
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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.
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Protein gel (SDS-PAGE) that has been stained with Coomassie Blue.
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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.
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Standard Western Blot
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