82316096-blot-ppt.pptx

BLOTTINGTECHNIQUES

Y . SunainaM . PharmacyIndustrial Pharmacy170211889011

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ELECTROPHORESIS

Electrophoresis - the migration of charged molecules in an electric field though a solution or solid support

We can separate DNA and RNA molecules by size using agarose gel electrophoresis

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DNA (or RNA) samples loaded into wells

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BLOTTING TECHNIQUES

Blotting – Transfer of DNA, RNA or Proteins, typically from a electrophoresis gel to a membrane e.g. nitrocellulose. This membrane can then be subject to further techniques such as hybridization.

Hybridization – Process where two

complementary single strands of nucleic acid (DNA or RNA) form a double helix.

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BLOTTING TECHNIQUES Southernblot for DNA analysis, requires electrophoresis

Northern blot for RNA analysis, requires electrophoresis

Western blot for protein analysis, requires electrophoresis

Dot blot: no electrophoresis required for DNA/RNA/protein analysis

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FLOW CHART OF SOUTERN BLOTTING

Preparing the samples and running the gel

Southern transfer

Probe preparation

Prehybridization

Hybridization

Post-hybridization washing

Signal detection

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SOUTHERN BLOTTING

Transfer buffer

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SOUTHERN BLOTTING

• Step 1: Digest the dsDNA– The DNA is isolated & digested with restriction

endonucleases.– Results in production of dsDNA fragments of varying

length depending on location of restriction sites.

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Step 2: Run the digest.Resulting fragments are separated

through agarose gel electrophoresis.

Standard DNA size markers should also be run - size of the sample fragments can be estimated

Stain with ethidium bromide & visualized under UV light.

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A depurination step is optional.DNA fragments larger than 15kb are

difficult to transfer HCl used to remove the purines,

reducing the size of the fragments

Step 3: Denature the DNA The agarose gel is soaked in a basic

solution, NaOH.After this , the base is neutralized

before proceeding to the membrane transfer

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Step 4: Transfer the denatured DNA to a filter membrane Nitrocellulose/nylon filter membranes

used. Nylon has higher binding capacity & less fragile.

In this, the DNA transfer is achieved by capillary action.Electrotransfer is less common.

After this, the filter membrane should be exposed to UV light to cross link the DNA fragments to the membrane.

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• Step 5: Hybridization AnalysisA) The membrane is soaked in a

prehybridization buffer. prevents nonspecific binding of the probe

B) It is then exposed hybridization probe—a single DNA fragment with a specific sequence.

• Contains a sequence that is complementary to the DNA sequence of interest.

• If the sequence of interest is present on the membrane, the probe will anneal to this sequence.

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Step 6 Post Hybridization

After hybridization, excess probe is washed from the membrane

The pattern of hybridization is visualized on X-ray film by autoradiography in the case of a radioactive or fluorescent probe, / by development of color if a chromogenic detection method is used.

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APPLICATIONS Diagnosis of HIV-1 infection Identify mutations, deletions, and gene

rearrangements In prognosis of cancer and in prenatal

diagnosis of genetic diseases

Investigation of diseases with less obvious genetic transmission, such as ischaemic heart disease

Malignant disease, hepatocellular carcinoma

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The main use is to identity any changes in DNA sequencing or genes expressed, e.g. comparing genes expressed by a diseased cell to genes of an healthy cell.

Restriction Fragment Length Polymorphism (RFLP)

Polymorphism refers to the DNA sequence variation between individuals of a species.  If the sequence variation occurs at the restriction sites, it could result in RFLP. example is the RFLP due to globin gene mutation

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RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP)

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NORTHERN BLOTTING

RNA was found not to bind with nitrocellulose filter.

RNA bands are blot transferred from the formaldehyde agarose gel into chemically reactive paper.

An aminobenzyloxymethyl cellulose paper prepared from whatmann filter paper number-540 after a series of uncomplicated reactions, is diazotized.

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They are hybridized & detected by autoradiography.

  Hybrids are treated with SI nuclease and

RNA ase which digests the single stranded RNA / DNA probe.

Structure of mRNA is revealed to the

extent to which mRNA protects the nucleic acid probe.

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NORTHERN BLOTTING

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WESTERN BLOTTING Extraction of protein from transformed

cells Separation of protein by using SDS-

PAGE where SDS acts as solvent for electrophoresis.

Transfer of electrophoresed gel in a buffer at low temperature(400 c ) for half an hour.

Blotting of proteins into nitrocellulose filter paper

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Placing of whatmann filter paper on a cathode plate followed by stack of coarse filter

whatmann filter electrophoresed gel, nitrocellulose filter, whatmann filter paper, coarse filter stack, whatmann filter &anode

plate

Putting the complete set up in transfer tank containing sufficient transfer buffer

Application of an electric field cause the migration of proteins from gel to nitrocellulose filter

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WESTERN BLOTTING

Hybridisation using radiolabelled antibodies(I123-antibodies)

Washing of the nitrocellulose filter with a wash solution (Tris-bufferd saline + Tween 20)

Detection of hybridised sequences by autoradiography.

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WESTERN BLOTTING PROCEDURE

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DOT BLOTS AND SLOT BLOTS

Cloned /pure extracted DNAs to be tested are spotted adjacent to each other on a nitrocellulose membrane

DNA blots thus produced are immobilized and denatured so that they are bound on membrane as single stranded DNA blots

The membrane is then hybridized with a radioactively labelled probe (DNA or RNA). The dot representing sequence related to probe will light up by autoradiography

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ADVANTAGES

No steps involving digestion with enzyme, gel electrophoresis or transfer from gel to membrane are needed in this technique.

Therefore it is more convenient , when no restriction sites are needed to be studied.

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DOT BLOTS FROM 25 HORDEUM SPECIES, SHOWING DIFFERENCE IN ABUNDANCE OF SEQUENCE RELATED TO PROBE PSC 119.

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An apparatus is used for placing spots on the membrane, through slots made in this equipment.

Slot blots from genomic DNA of 10 Avena species, showing difference in abundance of sequences related to two different probes(pAvs 2, pAvs 6).

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Southern blotting

Northern blotting

Western blotting

Molecule detected

DNA (ds) mRNA (ss) Protein

Gel electrophoresis

Agarose gel Formaldehyde agarose gel

Polyacrylamide gel

Gel pretreatment

DepurinationDenaturation &Neutralization

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Blotting method

Capillary transfer

Capillary transfer

Electric transfer

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REFERENCES PK Gupta., Elements of biotechnology, rastogi publications,

Meerut,2005. (page no: 55-57)

R.C.Dubey., A text book of biotechnology, Rajendra Ravindra printers ltd, Newdelhi,2006.(page no: 127-128)

  S.P.Vyas., Pharmaceutical biochemistry, CBS

publishers ,Newdelhi,1998. (page no: 389)  U.Satyanarayana, U.chakrapani, Biochemistry,

Arunabhasen books & allied pvt. Ltd, 2007.(page no: 587-589)

  Robert k.Murray, Daryl k.Granner, Peter A.Mayer, Victor

W.Rodwell. Harper’s illustrated biochemistry, Lange medical publications,1998.(page no. 403-404)

http://en.wikipedia.org/wiki/Western_blot

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Thank you