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Blotting Technique K.K.Gupta Drosphila Genetic Lab Blotting Technique www.dgen1996.wordpress.com K.K.Gupta Drosphila Genetic Lab. Dept.of Zoology, VBU,Hazaribag It is molecular biology technique for transferring proteins, DNA or RNA, on a nitrocellulose, polyvinylidene fluoride (PVDF) or nylon membrane for study of molecules for ex to find out the homologous se DNA /protein molecules by allowing DNA-DNA,DNA- RNA,Protein –protein hybridization. This hybridization of the two molecules are done after transferring the separated molecules from the gel onto the blotting membrane and thus called blotting techniques . Blotting techniques are followed by autoradiography for visualization of any molecules and its function. Types of Blotting Name of Molecules Probe Application blotting separated and blotted Southern DNA DNA Detection of similar DNA in unrelated sample Western Protein Antibody Similar protein in the sample South Protein DNA Detection of DNA binding protein western Eastern Protein cholera toxin, Detection of post translational modification concanavalin, phosphomolybdate Far – Oligosaccha Lecitins/Antibody Protein linked to oligosaccharides Eastern rides Northern RNA RNA/C-DNA Detection of similar m RNA Southern blotting A Southern blot is a method routinely used in molecular biology for detection of a specific DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis- separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization. Western blotting - A Western blot is used for the detection of specific proteins in complex samples. Proteins are first separated by size using electrophoresis before being transferred to an appropriate blotting matrix (usually PVDF or nitrocellulose) and subsequent detection with antibodies. Far-Western blotting Similar to a Western blot, the Far-Western blot uses protein–protein interactions to detect the presence of a specific protein immobilized on a blotting matrix. Antibodies are then used to detect the presence of the protein–protein complex, making the Far- Western blot a specific case of the Western blot. Southwestern blotting -A Southwestern blot is based on Southern blotting and is used to identify and characterize DNA-binding proteins by their ability to bind to specific oligonucleotide probes.[2] The proteins are separated by gel electrophoresis and are subsequently transferred to nitrocellulose membranes similar to other types of blotting Eastern blotting- The Eastern blot is used for the detection of specific posttranslational modifications of proteins. Proteins are separated by gel electrophoresis before being transferred to a blotting matrix whereupon posttranslational modifications are detected by specific substrates (cholera toxin, concanavalin, phosphomolybdate, etc.) or antibodies Far-Eastern blotting- The Far-Eastern blot is for the detection of lipid- linked oligosaccharides. High performance thin layer chromatography is first used to separate the lipids by physical and chemical characteristics, then transferred to a blotting matrix before the oligosaccharides are detected by a specific binding protein (i.e. antibodies or lectins). Northern blot The Northern blot is for the detection of specific RNA sequences in complex samples. Northern blotting first separates samples by size via gel electrophoresis before they are transferred to a blotting matrix and detected with labeled RNA probes. Reverse Northern blotting- The Reverse Northern blot differs from both Northern and Southern blotting in that DNA is first immobilized on a blotting matrix and specific sequences are detected with labeled RNA probes. Far-Eastern blotting- The Far-Eastern blot is for the detection of lipid- linked oligosaccharides. High performance thin layer chromatography is first used to separate the lipids by physical and chemical characteristics, then transferred to a blotting matrix before the oligosaccharides are detected by a specific binding protein (i.e. antibodies or lectins). Northern blot- The Northern blot is for the detection of specific RNA sequences in complex samples. Northern blotting first separates samples by size via gel electrophoresis before they are transferred to a blotting matrix and detected with labeled RNA probes Dot blotting- A Dot blot is a special case of any of the above blots where the analyte is added directly to the blotting matrix (and appears as a "dot") as opposed to separating the sample by electrophoresis prior to blotting. Comparison of blotting S,B NB WB Molecules detected DNA (ds) RNA(ss) Protein Gel electrophoresis Agarose gel Formaldehyde Agarose Polyacrylamide Gel gel Gel pre-treatment De-purination - - Denatyration and neutralization Blotting method Capillary transfer Capillary transfer Electric Transfer probes Radioactive labeled c-DNA, c-RNA Primary antibody DNA /Fluorescent radioactive labeled /nonradioactive Detection System Autoradiography Autoradigraphy Chemiluminescent Chemiluminescent Chemiluminescent Colometric Colometric Colometric Southern Blotting – Named after discover Edwin Southern . This is applied to transfer DNA molecules for the detection of unknown DNA fragments /sequence in a given sample . The method utilizes 1. Isolation of DNA 2. Restriction fragmentation of DNA 3. Separation of RF on Agarose Gel electrophoresis 4. Blotting on Nitocellulose membrane by capillary force . For this an arrangement is made in which a transfer buffer is poured in a glass tankand sponge block is kept over it . The the Gel with separated DNA fragment bands is kept and over it nitrocellulose membrane is put . The many folded blotting papers are kept and finally a weight of 0.5 kg is applied. 5. This allows the blotting procedure by capillary action and draws the buffer upward into the paper towels .As the buffer moves through the agarose gel ,it dissolves the DNA fragments and transferred on the surface of adjacent membrane . The pore size of blotting paper prevent its movement over the blotting paper . Membrane get imprinted with the DNA bands . 6. Treatment of Nitrocellulose membrane – The Nitrocellulose membrane with imprinted bands are the heated at 80oC in Vacuum to get the firm adherence of the DNA molecules 7. Incubation of the Nitrocellulose with transferred bands radioactive labeled probe . The probe is labeled by nick translation. A probe is first treated with trace amount Dnase which produces a nick in the DS DNA . The preparation is then incubated with DNA POL-I-an enzyme that posses both polymerase and exo-nuclease activities in the presence of labeled nucleotide. The DNA Pol will attach nick at which it will remove nucleotide towards 3’end and will add complimentary radio-labeled nucleotide in 5’-3’direction. This method is called nick translation. Fig. Probe Labelling by nick Translation Blotting Procedure Application Nucleic Acid Hybridization for Molecular Phylogeny Identification of Gene in the sample Detection of Post-translation modification in the protein Detection of antigenic protein Detection of DNA binding protein Detection of m RNA .
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