Ashwini blotting tech seminar

Ashwini blotting tech seminar

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  BLOTTING TECHNIQUES Presented by:Ashwini Patil M. Pharm (Pharmacology) Sem: II Department of Pharmacology R.C Patel Institute of Pharmaceutical Education and Research, Shirpur
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   Southern blottingis named after its inventor, the British biologist Edwin Southern (1975)  Other blotting methods Western (WB), Northern (NB) that employs similar principles, but using probes or RNA, have later been named in reference to Edwin Southern name 1
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   Visualisation ofspecific DNA ,RNA & Protein among many thousands of contaminating molecules requires no. of techniques which are collectively termed BLOT transfer and the process is called as Blotting.  A blot, in molecular biology and genetics, is a method of transferring proteins, DNA or RNA, onto a carrier  (for example, a nitrocellulose, polyvinylidene fluoride (PVDF) or nylon membrane) 2
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  Southern blotting Western blotting Northern blotting 3 1)Far westernblot 2)Far eastern blot 3)Reverse northern blot 4)Dot blot
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   PRINCIPLE:-  Thekey to this method is Hybridization.  Hybridization:-It is a process of forming a double stranded DNA molecule between a single stranded DNA probe and single stranded target DNA.  There are two important features of Hybridization i.e 1)The reactions are specific-the probes will bind to targets with complementary sequence. 2)The probe can find one molecule of target in a mixture of millions of related but non complementary molecules. 4
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   To identifyspecific DNA in a DNA sample.  To isolate desired DNA for construction of DNA.  To identify mutations, deletions, and gene rearrangements.  Used in prognosis of cancer and in prenatal diagnosis of genetic disease.  Used in phylogenetic analysis.  Diagnosis of HIV-1 and infectious disease.  In DNA fingerprinting 1) Paternity and maternity testing 2) Criminal identification and forensics 3)Personal identification 8
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   Northern blottingis a technique for detection of specific RNA sequences. Northern blotting was developed by James Alwine and George Stark at Stanford University (1979) and was named such by analogy to Southern blotting.  PRINCIPLE:- 1)Hybridization 2) Electrophoresis 3) Capillary action 9
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  Extract and purifymRNA from cells. Separated by gel electrophoresis Transfer to aminobenzyloxymethyl filter paper.(BLOTTING) Add labelled DNA probe for hybridization to take place Wash off unbound probe autoradiograph to detect mRNA- DNA hybrid. 10
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   Detecting aspecific mRNA in a sample.  Used in the screening of recombinants by detecting the mRNA produced by transgenesis.  In disease diagnosis.  In gene expression studies.  mRNA splicing studies.  Study RNA degradation.  Study RNA half life.  Time consuming procedure.  RNA samples can be degraded by RNAases.  Use of radioactive probes.  Detection of multiple probe is a problem. 12
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   The reversenorthern blot is a method by which gene expression patterns may be analyzed by comparing isolated RNA molecules from a tester sample to samples in a control cDNA library. 13
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   Quantification ofmRNA expression levels  Confirmation of differential display results  DNA Microarrays. Research Applications:- 1)Reverse northern blotting was used in a 2013 study in Gene in which the author identified a number of genes responsible for early cold-resistance response in the cold-hardy citrus fruit Poncirus trifoliata. 2)A study utilized the technique to determine differences in striatal tissue in rats treated with 3-NP, which is often used in experiments to generate a Huntington’s Disease-like phenotype in rats. 14
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  1 • cDNA sequencesfor transcripts of interest are immobilized on nylon membranes 2 • Prepared reverse northern blot membranes are pre- hybridized in Denhardt’s solution with SSC buffer and labeled cDNA probes are denatured at 100 °C and added to the pre-hybridization solution. 3 • The membrane is incubated with the probes for at least 15 hours at 65 °C, then washed and exposed.[3] 15
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   Western blottinguses specific antibodies to identify proteins that have been separated based on size by gel electrophoresis. The immunoassay uses a membrane made of nitrocellulose or PVDF (polyvinylidene fluoride).  The gel is placed next to the membrane and application of an electrical current induces the proteins to migrate from the gel to the membrane.  The membrane can then be further processed with antibodies specific for the target of interest, and visualized using secondary antibodies and detection reagents. 17
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   Principle:-  Itis based on the principle of immunochromatography.  Antigens are separated by Poly Acrylomide Gel Electrophoresis(PAGE).  Antibodies in serum react with specific antigens.  Signals are detected according to the principles of test systems. 18
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   The technique usesthree elements (1) separation by size (2) Transfer to a solid support (3) Marking target protein using a proper primary and secondary antibody to visualize. 19
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  Thermo Scientific myECLImager 20
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   Used topinpoint a specific protein in a given sample, employs the ability of an enzyme or fluorescence-labeled primary antibody to bind to its specific antigen.  Sensitivity:-Its ability to detect as little as 0.1 nanograms of protein in a sample.  Fewer antibodies are needed for testing, which cuts down laboratory costs significantly.  Specificity:-specific antibodies show affinity for specific proteins, the process can selectively detect a target protein even in a mixture of 3 lakh different proteins. 22
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   Prone toFalse or Subjective Results:- A false- positive results comes when an antibody reacts with a non-intended protein, which is what frequently happens when a patient being tested for HIV.  On the other hand false result can easily occur if larger proteins are not given sufficient time to transfer properly to the membrane.  High Cost and Technical Demand:-A delicate process, western blotting requires precision in every step for proper identification of a sample's constituents. 23
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   Highly sensitivemethod to detect a specific protein even in very low quantity.  Used in clinical diagnosis  Quantifying a gene product (gene expression studies)  Some forms of Lyme disease testing employ Western blotting . 24
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  To identify proteinsthat interact with a given protein. Far-Western blot analysis is an alternative method to analyze protein-protein interactions. PRINCIPLE:- The principle of far-western blotting is modified from western blotting. 25
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  1 • Uses thenatively-folded recombinant protein as the first probe to interact with proteins that have been separated on a PVDF membrane. 2 • The second probe used in this platform is the specific antibody against the original protein, while the HRP- conjugated third probe is the antibody against the second probe. 3 • When combined with protein identification using LC- MS-MS, the resulting protein spots (or protein bands) can be characterized and then subjected to further functional assays, such as protein immuno-precipitation. 26
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   Low experimentalcost.  High sensitivity. 27
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   Far-Eastern blottingis a technique developed in 1994 by Taki and colleagues at the Tokyo Medical and Dental University, Japan for the analysis of lipids separated by high- performance thin layer chromatography.  The lipids are transferred from the HPTLC plate to a PVDF membrane for further analysis, for example by enzymatic or ligand binding assays or mass spectrometry. 28
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   Purification ofphospholipids.  Structural analysis of lipids in conjunction with direct mass spectrometry.  Binding study using various ligands such as antibodies, lectins, bacterium, viruses, and toxins.  Enzyme reaction on membranes. 30
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   A techniquefor detecting, analyzing and identifying proteins, similar to the western blot technique but differing in that protein samples are not separated electrophoretically but are spotted through circular templates directly onto the membrane or paper substrate. 31
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   Estimation ofconcentration of proteins in crude preparations (such as culture supernatant).  The size of complementary sequences is obtained through the process. 33
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   http://ourpastimes.com/advantages- disadvantages-western-blot-8670663.html  http://www.biologyexams4u.com/2014/01/weste rn-blotting-principle-summary-of.html https://www.researchgate.net/figure/231212350_fi g1_Figure-1-Schematic-diagram-of- immunochromatography  http://www.biologyexams4u.com/2013/12/south ern-blotting-procedure-steps.html  http://i-base.info/guides/testing/test-accuracy- results-and-further-testing  http://www.aidsmap.com/Confirmatory- tests/page/1323369/ 34
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   http://labtestsonline.org.uk/understanding/a nalytes/lyme/tab/test/  http://www.drzeegers.com/2014/08/lyme- disease-diagnosis-blood-test-tickborne-disease/ http://www.biologyexams4u.com/2014/01/n orthern-blotting-principle-summary-of.html  Sritunyalucksana K, Wannapapho W, Lo CF, Flegel TW. (2006) PmRab7 is a VP28-binding protein involved in white spot syndrome virus infection in shrimp. J Virol 80: 10734-10742  www.scribd.com  http://webap.rsh.ncku.edu.tw/shrimpwssv/in dex.php/sfgc-technological-platforms/protein- interactions-using-far-western-blotting 35
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   http://www.wikivisually.com/wiki/Reverse_N orthern_blot  http://www.abcam.com/protocols/dot-blot- protocol http://feedforbiotech.blogspot.com/2011/03/dot- blot.html  https://www.thermofisher.com (far western)  https://fr.wikipedia.org/wiki/Far-eastern_blot  https://www.pathwayz.org/Tree/Plain/TRANS GENESIS  https://openi.nlm.nih.gov(mrna degradation) 36
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   http://www.tiem.utk.edu/~gross/bioed/web modules/mRNA.htm  http://bitesizebio.com/7206/introduction-to- dna-microarrays/ 37