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Macromolecule Blotting

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Macromolecule Blotting

Description of Module

Subject Name

Paper Name

Module Name/Title Macromolecule Blotting

Dr. Vijaya Khader Dr. MC Varadaraj

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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

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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.

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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