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Macromolecule Blotting - e- · PDF fileMacromolecule Blotting Blotting Southern Northern...

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  • 1

    Macromolecule Blotting

  • 2

    Macromolecule Blotting

    Description of Module

    Subject Name

    Paper Name

    Module Name/Title Macromolecule Blotting

    Dr. Vijaya Khader Dr. MC Varadaraj

  • 3

    Macromolecule Blotting

    1. Objectives

    1. Blotting/Hybridization and its principle

    2. Types of Blotting

    3. Applications

    2. Lay Out

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    Macromolecule Blotting

    Blotting

    Southern Northern Western

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    Macromolecule Blotting

    3. 1 Description

    Blotting means to transfer DNA or RNA to nitrocellulose membrane and probe binds to

    complementary strand in the sample to give result in the form of signal using radioactive or biotin

    labeled probe.

    Principle

    At high temperature two strands of DNA get separated they and re-anneal when temperature is

    brought back to normal. There are 2 important features of hybridization:

    The reaction is very specific that means the probes will only bind to targets with a

    complementary sequence.

    The probe can identify one molecule of target in a mixture of millions of related and non-

    complementary molecules

    2 Types of Blotting

    2.1 Southern Blot:

    It is a method in molecular biology which is used for the identification of a specific DNA sequence

    in a mixture of DNA samples. In this DNA is first run on agarose gel and than transferred to

    membrane and further detection using specific probe. This method is known by its inventors name

    who was biologist by profession, Edwin Southern. After this other methods of blotting are named

    Northern and western further in continuation to Southern blot.

    2.2 Northern Blot:

    The northern blot is technique used in molecular biology research to study expression of by

    detecting RNA (or isolated mRNA) in a sample. Northern blotting involves the use of agarose gel

    electrophoresis to separate RNA samples by size, and detects with a hybridization probe which is

    complementary target sequence. Blot means to transfer RNA from the electrophoresis gel to the

    blotting membrane. This technique was given in 1977 by James Alwine, David Kemp, and George

    Stark at Stanford University.

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    Macromolecule Blotting

    2.3 Western Blot:

    The western blot is a widely used analytical technique used to detect specific proteins in a sample.

    It uses SDS to separate proteins. The proteins from the SDS gel are then transferred to a

    nitrocellulose or PVDF membrane, and incubated with antibodies specific to the target protein.

    2.4 Eastern Blot:

    To study post translational modification this technique is (lipids, phosphomoieties and

    glycoconjugates). It is mostly used to detect carbohydrate epitopes. Eastern blotting is

    considered an extension of the Western blotting. Transferred proteins are analyzed for post-

    translational modifications using specific probes that detect lipids, carbohydrate, phosphorylation

    or any other protein modification.

    3 Probe:

    Is defined sequence which is used to search mixtures of nucleic acids for molecules containing a

    complementary sequence. Any positive sample can be used as control (eg. In diagnosis of

    viruses)

    Blotting Membranes

    Membrane or support onto which the separated proteins are transferred which bind proteins with

    high affinity:

    Nitrocellulose membrane

    has very good protein binding capacity and retention ability

    Polyvinylidene fluoride (PVDF) membrane

    Its strength is more than nitrocellose membrane so it can be used if reprobing and stripping is

    required

    Summary of steps

    Run samples on a gel/make a slot/dot blot

    Transfer to a membrane

    Link sample to membrane

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    Macromolecule Blotting

    Make a probe

    Hybridize probe to membrane

    Detect probe

    Labeling of DNA or RNA probes

    Radioactive labeling: Use of radioactive material for labelling

    Non-radioactive labeling: Enzymatic labeling using biotin labeled probes

    End labeling: Labels at the ends

    Uniform labeling: labels internally

    Hexanucleotide primered labeling:

    After denaturing DNA add random hexamer primers and DNA polymerase

    Known viral gene sequence (or any other DNA/RNA ) run in 1% agarose gel. Viral DNA fragment is

    eluted from the gel and incubated in boiling water bath for 10 min, for denaturation of DNA

    Table: Probe preparation mix

    Denatured DNA 200-500 ng

    Random Primer 100 ng

    10x Klenow buffer 3 l

    dNTP mix (-CTP)(3.3nM each) 4.5 l

    -32P dCTP (10Ci/l,specific activity 3x103 Ci/mmole) 10 Curie

    Klenow enzyme 5 units

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    Macromolecule Blotting

    Final volume with ddH2O 30 l

    The reaction is incubated at 37C for one hour.

    To this, equal volume of Buffer-A is added.

    The mixture is then denatured by incubating the tube in boiling water bath for 7 min.

    The contents are immediately transferred to ice before adding to the hybridization bottle.

    dNTP mix: (for 32-P dCTP as the radioactive molecule, 100mM stock of dATP, dTTP and dGTP):

    1+1+1+27l water. 4.5l of this mix is used for one reaction.

    Buffer-A: 500mM Tris HCL (pH 7.5), 500mM NaCl, 5mM EDTA and 0.5% SDS.

    Blotting in virus diagnostics(Southern and Northern)

    Procedure for northern and Southern blot is same except that in Southern DNA is used and in

    Northern RNA is used.

    Probes prepared from cloned viral cDNAs are used in Southern blots to detect the viral DNA in

    infected plants.

    A modification in this technique called tissue printing employs blotting of leaf squashes rather

    than the viral DNA on to nylon membranes .

    Use of radioactive material for preparing the probes has been the major limitation of this

    technique.

    To avoid this hazardous material, non-radioactive labelling techniques have been developed

    6.1 Diagnosis of RNA and DNA viruses

    Slot-Blot Hybridization

    Samples are crushed in DNA/RNA isolation buffer and centrifuged to remove debris (CTAB Buffer

    or TRI reagent etc.)

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    Macromolecule Blotting

    Approx 200l liquid sample is denatured in a boiling water bath for 5 min before loading.

    The slot-blot manifold is assembled with positively charged nylon membrane, previously wetted

    with sterile water.

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    Macromolecule Blotting

    Slot -blot and dot-blot

    apparatus

  • 11

    Macromolecule Blotting

    Procedure

    The manifold is connected to vacuum pump.

    To each well, 10x SSC buffer (200l) is added and vacuum was applied till the buffer is completely

    absorbed but not dried.

    After that, 200l each of the denatured samples is loaded to the wells

    Vacuum is applied till the samples is absorbed completely.

    Afterwards, 200l of 10x SSC is added and allowed to completely pass through.

    20x SSC Buffer: 3M NaCl and 0.3 M Trisodium citrate

    Vacuum is released, apparatus disassembled and the membrane is rinsed in 2x SSC.

    The membrane is air dried and exposed to UV light for 2 min in a UV crosslinker for binding of the

    transferred nucleic acids.

    The membrane is wrapped in saran film and stored at 4C until hybridization.

    After completion of electrophoresis, the DNA in the gel is denatured by placing the gel in a plastic tray

    containing the denaturation solution with slow shaking on a gel rocker for 30 min.

    After denaturation, the gel is soaked in neutralizing solution for 30 min with slow shaking.

    The two chambers of the electrophoresis tank are filled with transfer solution (10x SSC).

    A wick of Whatman 3MM paper is soaked in 10x SSC and placed over the gel platform of the tank with

    its ends submerged in the solution.

    Figure: Membrane after dot-blot transfer

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    Macromolecule Blotting

    Denaturing Solution: 1M NaCl and 0.5M NaOH

    Neutralizing Solution: 1.5M Tris HCl (pH 8) and 3M NaCl

    Three more sheets of Whatman 3MM paper (slightly bigger than the gel) are soaked in 10x SSC and

    placed over the wick.

    The gel is carefully inverted and placed over the sheets avoiding any air bubbles between the gel and

    Whatman papers.

    Nylon membrane is cut to the gel size, wetted in sterile water and placed over the gel avoiding any air

    bubbles.

    Three more sheets of Whatman 3MM paper are cut to gel size, soaked in 10x SSC and placed over the

    membrane.

    A stack of gel-sized dry blotting papers (8-10 cm height) are placed over the 3MM papers and

    approximately 500 g weight is placed on top of the stack.

    The transfer is allowed to take place for 14-16 h.

  • 13

    Macromolecule Blotting

    After completion of the transfer, the blotting paper stack is removed, the membrane is marked so as to

    indicate the orientation and rinsed in 2x

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