BLOTTING TECHNIQUESBLOTTING TECHNIQUESM.Prasad NaiduMSc Medical Biochemistry, Ph.D,.

Definition 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 Types of blotting techniquestechniques

1 ) Southern blotting ( to detect DNA )

2 ) Northern blotting ( to detect RNA )

3 ) Western blotting ( to detect protein )

Southern BlottingSouthern BlottingIn 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 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 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 .

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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 .

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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.

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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 .

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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 .

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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.

BlottingBlottingTransfer 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 by2) 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 .

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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 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 .

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 .

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 .

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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 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 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 .

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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 .

ApplicationsApplicationsA 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 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

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 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 .

contdcontdBlot 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 .

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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 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 .

contdcontdThe 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 .