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Blotting techniques
Blotting techniques
Blotting techniques
Blotting techniques
Blotting techniques
Blotting techniques
Blotting techniques
Blotting techniques
Blotting techniques
Blotting techniques
Blotting techniques
Blotting techniques
Blotting techniques
Blotting techniques
Blotting techniques
Blotting techniques
Blotting techniques
Blotting techniques
Blotting techniques
Blotting techniques
Blotting techniques
Blotting techniques
Blotting techniques
Blotting techniques
Blotting techniques
Blotting techniques
Blotting techniques
Blotting techniques
Blotting techniques
Blotting techniques
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Blotting techniques

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    • 1. BLOTTINGTECHNIQUESBLOTTINGTECHNIQUES M.Prasad Naidu MSc Medical Biochemistry, Ph.D,.
    • 2. DefinitionDefinition Visualization of specific DNA , RNA & protein among many thousands of contaminating molecules requires the convergence of number of techniques which are collectively termed BLOT transfer .
    • 3. Types of blotting techniquesTypes of blotting techniques 1 ) Southern blotting ( to detect DNA ) 2 ) Northern blotting ( to detect RNA ) 3 ) Western blotting ( to detect protein )
    • 4. Southern BlottingSouthern Blotting In 1975 Edward Southern developed this technique that is widely used to detect fragments of DNA .  This requires 1 ) Separation of DNA or DNA fragments by agarose gel electrophoresis . 2 ) DNA fragments are blotted onto a strip of nitrocellulose or a nylon membrane. 3 ) Identification by hybridization with a labeled ,complementary nucleic acid probe.
    • 5. DefinitionDefinition Southern blot a method for transferring DNA from an agarose gel to nitrocellulose filter , on which the DNA can be detected by suitable probe ( eg : complementary DNA or RNA ) .
    • 6. ProcedureProcedure The DNA sample is digested by restriction endonucleases , producing small fragments & that are amenable for analysis . Fragments are seperated by agarose gel electrophoresis or PAGE . The mobility of nucleic acids in agarose gels is influenced by agarose concentration , molecular size & molecular conformation of the nucleic acid .
    • 7. Agarose concentration of 0.3 – 2 %are most effective for nucleic acid separation . Like proteins nucleic acids migrate at rate that is inversely proportional to the logarithm of their molecular weight .
    • 8. contdcontd Separated nucleic acids are visualized by fluorescent dye ethidium bromide . The agarose gel is soaked in a solution of dye & washed for remain excess dye . illumination of the rinsed slab with UV light reveals red orange stains where nucleic acids are located .
    • 9. contdcontd Ethidium bromide stains both single & double stranded nucleic acids , the fluorescence is much greater with double stranded molecules . The electrophoresis can be performed with dye incorporated in the gel & buffer . This has the advantage that the gel can be illuminated with UV light during electrophoresis to view the extent of separation.
    • 10. contdcontd The mobility of DNA may be reduced by 10 -15 % in the presence of ethidium bromide . Ethidium bromide must be used with great care as it is a potent mutagen . Gloves should be worn at all times while using the dye solutions or handling gels .
    • 11. contdcontd Newer fluorescent SYBR dyes produced by molecular probes offer several advantages , less toxic & 5 times more sensitive than ethidium bromide. Labeled DNA with radioisotope P32 at 5’ & 3’ ends . P 32 is a strong β emitter . Bands of labeled DNA on electrophoresis gel can located by autoradiography .
    • 12. contdcontd Labelling molecule before analysis with coenzyme biotin , biotin forms a strong complex with enzyme linked streptavidin . PAGE is useful for analysing small fragments of DNA upto 3,50,00 daltons ( 500 bp ) in molecular size . Large molecules of DNA could be separated by pulsed field gel electrophoresis.
    • 13. BlottingBlotting Transfer of DNA from gel to nitrocellulose membrane done by 1 ) Weak acid treatment to depurinate &fragment the DNA , thus make it smaller & easier to elute from the gel . followed by 2) Denaturate with strong base& neutralisation ( hydrolyzes phosphodiester back bone at depurinated sites ) single strands bind to membranes more efficiently ) A buffer is used to facilitate the transfer .
    • 14. contdcontd Original methods of transfer relied on capillary action . Vaccum or preassure systems can be used to speed the transfer . Faster & more efficient transfer is afforded by the use of an electroblotter . Electroblotting process is usually completes in 1-4 hours .
    • 15. Hybridization assaysHybridization assays All hybridization assays are based on the ability of nucleic acids to form specific double stranded hybrids . The process requires 1 ) A probe that can target nucleic acids & allow for specific complemenatary base pairing . 2) A method to detect any resulting double strands nucleic acids .
    • 16. contdcontd  Conditions of high stringency in hybridization assay are 1) Low salt concentration , 2) High formamide levels , 3) High temparature . As the stringency of the assay is lowered increasing number of base mismatches are tolerated . conditions of high stringency require exact base pairing .
    • 17. The time required to hybridize the probe to a given fraction of the target remains proportional to the probe concentration . The rate of hybridization reaction is influenced by temperature & ionic strength. Above the Tm no stable hybrids are present . Divalent cations like Mg+2 have stronger effect on hybridization .
    • 18. contdcontd Unbound probes are removed by washing Probe bound to the membrane is detected by autoradiogarphy , which reveals the DNA fragments to which the probe hybridized .
    • 19. ApplicationsApplications Southern blots are used in gene discovery, mapping , evolution & development studies , diagnostics & forensics . Deletions / insertions . pointmutations / polymorphisms . Structural rearrangements . Allow for determination of molecular weights of restriction fragments . Presence of particular bit of DNA in the sample.
    • 20. Northern blottingNorthern blotting Northern blotting is a technique for detection of specific RNA sequences . Developed by James alwine & George stark. RNA molecules have defined length & much shorter than genomic DNA it is not necessary to cleave RNA before electrophoresis . RNA is more susceptible to degradation than DNA . RNA sample are separated based on size by gel electrophoresis .
    • 21. contdcontd RNA is blotted on to a nylon positively charged membrane . The membrane is placed in a hybridization buffer with a labeled probe ( usually DNA ) Labeled probe is detected by autoradiography Expression patterns of sequences of interest in different samples can be compared .
    • 22. ApplicationsApplications A standard for direct study of the gene expression at the level of mRNA . Detection of mRNA transcript size . Study of RNA splicing – can detect alternatively spliced transcripts . Study RNA half life
    • 23. DisadvantagesDisadvantages Time consuming procedure . RNA samples can be degraded by RNases . Use of radioactive probes . Detection with multiple probes is a problem .
    • 24. Western blottingWestern blotting Western blotting is an immunoblotting technique which rely on the specificity of binding between the molecule of interest & a probe to allow detection of molecule of interest in a mixture of many other similar molecules . In western blotting the molecule of interest is a protein & the probe is typically an antibody raised against that particular protein .
    • 25. ContdContd SDS PAGE technique is a prerequisite for western blotting . Protein sample is subjected to electrophoresis on SDS polyacrylamide gel . Electroblotting transfers the separated proteins from the gel to the surface of nitrocellulose membrane .
    • 26. contdcontd Blot is incubated with generic protein ( such as milk protein )which binds to any remaining sticky places on the nitrocellulose . An antibody which is specific for the protein of interest ( the primary antibody Ab 1 ) is added to the nitrocellulose sheet & reacts with the antigen . Only the band containing protein of interest binds the antibody forming a layer of antibody molecules .
    • 27. contdcontd Following several rinses for removal of nonspecifically bound Ab1 , the Ab1 – antigen complex on the nitrocellulose sheet is incubated with second antibody Ab2 , which specifically recognizes the Fc domain of the primary antibody & binds it . Ab 2 is radioactive labeled or is covalently linked to reporter enzyme which allows to visualize protein – Ab1 – Ab2 complex .
    • 28. ApplicationsApplications The confirmatory HIV test employs a western blot to detect anti HIV antibody in a human sample . Proteins from known HIV infected cells are separated & blotted on a membrane then the serum to be tested is applied in the primary antibody incubation step. Free antibody is washed away & a second anti human antibody linked to an enzyme signal can be added .
    • 29. contdcontd The stained bands then indicate the proteins to which the patient serum contains antibody . Western blot is also used as definitive test for bovine spongiform encephalopathy . ( mad cow disease ) Some forms of Lyme disease testing employs western blotting .

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