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BLOTTING TECHNIQUES M.Prasad Naidu MSc Medical Biochemistry, Ph.D,.
Definition • 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 .
Types of blotting techniques • 1 ) Southern blotting ( to detect DNA ) • 2 ) Northern blotting ( to detect RNA ) • 3 ) Western blotting ( to detect protein )
Southern 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.
Definition • 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 ) .
Procedure • 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 .
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 .
contd • 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 .
contd • 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.
contd • 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 .
contd • 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 .
contd • 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.
Blotting • 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 .
contd • 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 .
Hybridization 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 .
contd • Conditions of high stringency in hybridization assay are • Low salt concentration , • High formamide levels , • 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 .
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 .
contd • 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 .
Applications • 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.
Northern 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 .
contd • 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 .
Applications • 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
Disadvantages • Time consuming procedure . • RNA samples can be degraded by RNases . • Use of radioactive probes . • Detection with multiple probes is a problem .
Western 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 .
Contd • 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 .
contd • 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 .
contd • 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 .
Applications • 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 .
contd • 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 .
