Blotting techniques


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  • Blotting techniques

    1. 1. BLOTTINGTECHNIQUESBLOTTINGTECHNIQUES M.Prasad Naidu MSc Medical Biochemistry, Ph.D,.
    2. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 23. DisadvantagesDisadvantages Time consuming procedure . RNA samples can be degraded by RNases . Use of radioactive probes . Detection with multiple probes is a problem .
    24. 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. 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. 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. 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. 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. 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 .