BLOTTING TECHNIQUES

Present by…

CHOVATIYA MANISH M.M.SC BIOTECHNOLOGY

Blots are techniques for transferring DNA , RNA

and proteins onto a carrier so they can be

separated, and follows the use of a gel

electrophoresis. The Southern blot is used for

transferring DNA, the Northern blot for RNA and

the western blot for PROTEIN.

Blotting technique

Southern Blot

It is used to detect DNA.

Northern Blot

It is used to detect RNA.

Western blot

It is used to detect

protein.

Professor Sir Edwin Southern,Professor of Biochemistry andFellow of Trinity developed thismethod in 1975.

Southern won the LaskerAward for Clinical MedicalResearch prize for the methodof finding specific DNAsequences he developed thisprocedure at Edinburgh

University more than 30 yearsago. The technique is knownas DNA transfer or 'Southernblotting'

Professor Sir Edwin Southern

This method Involves separation, transfer and

hybridization.

It is a method routinely used in molecular biology for

detection of a specific DNA sequence in DNA samples.

The DNA detected can be a single gene, or it can be part of

a larger piece of DNA such as a viral genome.

Southern blotting combines agarose gel electrophoresis for

size separation of DNA with methods to transfer the size

separated DNA to a filter membrane for probe

hybridization.

The key to this method is Hybridization.

Hybridization - Process of forming a double-stranded

DNA molecule between a single-stranded DNA probe and a

single-stranded target patient DNA.

1. The mixture of molecules is separated.

2. The molecules are immobilized on a MEMBRANE.

3. The probe is added to the membrane to bind to

the molecules.

4. Any unbound probes are then removed.

5. The place where the probe is connected corresponds to

the location of the immobilized target molecule.

Cellulose nitrate (nitrocellulose)

It is produced by treating cellulose withnitric acid.

Each glucose unit in the cellulose polymer isesterified with three nitrate groups, andthese nitrate groups are responsible for boththe negative charge of nitrocellulose atneutral pH and the unusual flammability ofdry nitrocellulose.

The major improvement has been the introduction of nylon membranes,

which have three advantages over their nitrocellulose counterparts.

First,nylon membranes are less fragile than nitrocellulose sheets,the latter

tending to crack if handled roughly during Southern blotting,and usually

disintegrating if attempts are made to carry out more than two or three

hybridization analyses with the same blot.

Nylon membranes cannot be damaged by handling and a single blot

can be rehybridized up to ten times,this limit being due not to eventual

breakage of the membrane but to the gradual loss of the blotted DNA

during repeated hybridizations.

The second advantage of nylon membranes is that under certain

conditions (a positively charged membrane and an alkaline transferbuffer) the transferred DNA becomes covalently bound to the membrane

during the transfer process. This is not the case with a nitrocellulose

membrane,which initially binds DNA in a semipermanent

manner,immobilization occurring only when the membrane is baked at

80C.

Transfer onto a positively charged nylon membranecan therefore reduce the possible loss of DNA thatmight occur by leaching through the membraneduring the blotting process;

it is also quicker,the transfer time being reduced from18 h to 2 h.

Finally,nylon membranes efficiently bind DNAfragments down to 50 bp in length,whereasnitrocellulose membranes are effective only withmolecules longer than 500 bp.

Nitrocellulose has not,however,been completelysuperseded because it has one significantadvantage compared with nylon membranes: areduced amount of backgroundhybridization,especially with probes that have beenlabelled with nonradioactive markers.

1. Digest the DNA with an

appropriate restriction

enzyme.

2.The complex mixture of

fragments is subjected to

gel electrophoresis to

separate the fragments

according to size.

The restriction fragments present in the gel are denatured with alkali

TO GET ss DNA (break Hydrogen bond).

To break the DNA molecules in individual bands within the gel into smaller

fragments, because smaller fragments transfer more quickly than larger

ones. This is achieved by soaking the gel in 0.25 molL21 HCl for 30

min,which results in a small amount of depurination – cleavage of the

b-N-glycosidic bond between purine bases (adenine or guanine) and

the sugar component of their nucleotides – which is followed by

decomposition of the sugar structure and breakage of the

polynucleotide chain.

If a nitrocellulose membrane is being

used then the alkali pretreatment is

followed by neutralization of the gel by

soaking in a Tris-salt buffer,this step being

essential because DNA does not bind to

nitrocellulose at a pH of greater than 9.0.

Nitrocellulose membrane soaked in salt solutionis placed over gel. Nucleic acid move from gelto membrane . a high concentration of salt(NaCl THAT IS the positive Na+ ions shield thenegative charges on the phosphodiesterbackbone AND LOWERS –ve DNA = -vemembrane repelling each other

BAKING Leads to fixing of DNA strongly to thenitocellulose membrane. UV irradiation,whichresults in covalent attachment of DNA to anylon membrane.

3 METHODS OF TRANSFER : CAPILLARY

In a capillary system, rate of transfer depends on the size ofthe DNA, thickness of the gel, and agarose concentration.

Upward capillary transfer is slow, the architecture of the blotcrushes the gel and retards diffusion of the DNA. With a high-salt buffer, it takes appr. 18 hrs to obtain acceptable transferof a 15 kb molecule from a 5 mm thick 0.7% agarose gel; withthe same gel 90% of the 1 kb molecules will be transferred in 2hrs. This problem is partially alleviated by the depurinationstep, which breaks larger molecules in to fragments 1 to 2 kbin length, thereby reducing the time needed for their transfer.If the gel is thicker than 5 mm or has an agaroseconcentration > 1 %, it cannot be assumed that the largerfragments will have transferred to a sufficient extent after 12hr.

Capillary blotting apparatus

The weight

helps only in

bringing all

surfaces

together. High

wt may break

the gel.

First,the membrane is prehybridized in a solution designed toblock the unused DNA binding sites on the membranesurface. If this step is omitted then the probe will bindnonspecifically to the surface of the membrane and thesignal resulting from hybridization to the specific restrictionfragment will be difficult if not impossible to identify.

The prehybridization solution therefore contains nonbiologicalpolymeric compounds such as polyvinylpyrrolidone and/orbiological polymers such as Ficoll (a carbohydrate-basedcompound),bovine serum albumin or dried milk. DNA froman organism unrelated to the one whose DNA has beenblotted can also be used (salmon sperm DNA is a popularchoice).

Prehybridization takes between 15 min and 3 h at68C,depending on the type of membrane

enough labeled probe DNA is added to hybridize to the target restriction fragment to produce a clear signal that can be discerned by the detection system appropriate for the label carried by the probe.

The second critical factor that must be considered during thehybridization step is the specificity of the reaction. If theprobe DNA has been carefully chosen then it will contain aregion that is completely complementary to all or a part ofthe blotted restriction fragment that is being sought. If thishybridizing region in the probe is not completelycomplementary to the target,then it will at least have aregion of strong similarity so that a stable hybrid can form.The problem is that the probe also has the potential tohybridize to any other blotted DNA fragments with which ithas partial complementarity.

Temperature is relevant because the meltingtemperature (Tm,the highest temperature atwhich the hybrid is stable) of a fully base-paired hybrid is higher than that for one inwhich some base pairs have not formedbecause the probe and target DNAs are notfully complementary. Hybridization Maximumrate occurs at 20-25°C below the Tm for DNA-DNA hybrids, 10-15°C below Tm for DNA-RNAhybrids

Formation of the desired hybrid,anddestabilization of nonspecific hybrids,cantherefore be achieved by utilizing anappropriate combination of buffer compositionand hybridization temperature.

The probe hybridizes to the

complementary DNA restriction

fragment.

Excess probe is washed away

and the probe bound to the filter is

detected by autoradiography,

which reveals the DNA fragment

to which the probe hybridized.

Southern blots are used in gene discovery , mapping,

evolution and development studies, diagnostics and

forensics (It is used for DNA fingerprinting, preparation of

RFLP maps)

identification of the transferred genes in transgenic

individuals, etc.

Southern blots allow investigators to determine the

molecular weight of a restriction fragment and to measure

relative amounts in different samples.

Southern blot is used to detect the presence of a particular

bit of DNA in a sample

analyze the genetic patterns which appear in a person's

DNA.

Northern blotting is a technique for detection of specific

RNA sequences.

Northern blotting was developed by James Alwine and

George Stark at Stanford University (1979) and was

named such by analogy to Southern blotting

The first is to determine which tissues

express a particular gene, and this can

give some indication of the physiological

function of the encoded protein.

The second principal reason for measuring

an mRNA is to determine the factors which

regulate the expression of a given gene,

be they nutritional, hormonal, or environmental.

Northern blotting.

A second method is the RNase protection assay, which is generally considered to offer improved sensitivity.

The third method utilizes the reversetranscriptase polymerase chain reaction; thisprovides considerable increases in sensitivityover Northern blotting and the RNaseprotection assay, and may be useful formeasuring very low levels (a few copies) of anmRNA in a tissue.

Then, in a single step reaction, the

nucleases are inactivated and the

remaining probe:target hybrids are

precipitated. These products are

separated on a denaturing

polyacrylamide gel and are visualized by

autoradiography.

1. RNA is isolated from several biological

samples (e.g. various tissues, various

developmental stages of same tissue etc.)

1. Eukaryotic mRNA can then be isolated

through the use of oligo (dT)

cellulose chromatography to isolate only

those RNAs with a poly(A) tail

2.Sample’s are loaded on

gel and the RNA samples

are separated according

to their size on an

agarose gel .

The resulting gel following

after the electrophoresis

run.

3. The gel is then blotted on a

nylon membrane or a

nitrocellulose filter paper

by creating the

sandwich arrangement.

Nylon membrane with a

positive charge is the most

effective for use in northern

blotting since the negatively

charged nucleic acids have

a high affinity for them.

Once the RNA has been transferred to the membrane, it is immobilized through covalent linkage to the membrane by UV light or heat.

4. The membrane is placed in a dish

containing hybridization buffer

with a labeled probe.

Thus, it will hybridize to the RNA

on the blot that corresponds to

the sequence of interest.

5. The membrane is washed to

remove unbound probe.

6. The labeled probe is detected

via autoradiography or via a

chemiluminescence reaction (if

a chemically labeled probe is

used). In both cases this

results in the formation of a

dark band on an X-ray film.

Now the expression patterns of

the sequence of interest in the

different samples can be

compared.

The RNA samples are most commonly

separated on agarose gels

containing formaldehyde as a

denaturing agent for the RNA to limit

secondary structure.

Polyacrylamide gel electrophoeresis

with urea can also be used in RNA

separation but it is most commonly used

for fragmented RNA or microRNAs

A standard for the study of gene expression at the level of

mRNA (messenger RNA transcripts)

Detection of mRNA transcript size

Study RNA degradation

Study RNA splicing

Study RNA half-life

Often used to confirm and check transgenic / knockout

mice (animals)

Western blotting (1981) is an Immunoblotting technique

which rely on the specificity of binding between a protein

of interest and a probe (antibody raised against that

particular protein) to allow detection of the protein of

interest in a mixture of many other similar molecules.

Detects proteins and estimates their molecular weight.

Used to detect changes in protein expression.

Tissue preparation

Samples can be taken from whole tissue or from cell culture. Solid tissuesare first broken down mechanically using a blender (for larger samplevolumes), using a homogenizer (smaller volumes), or by sonication. Cellsmay also be broken open by one of the above mechanical methods.However, virus or environmental samples can be the source of proteinand thus western blotting is not restricted to cellular studies only.

Assorted detergents, salts, and buffers may be employed toencourage lysis of cells and to solubilize proteins. Protease andphosphatase inhibitors are often added to prevent the digestion of thesample by its own enzymes. Tissue preparation is often done at coldtemperatures to avoid protein denaturing and degradation.

A combination of biochemical and mechanical techniques –comprising various types of filtration and centrifugation – can be used toseparate different cell compartments and organelles.

1. After the samples have been prepared, they are separated

by size using SDS-PAGE (sodium dodecyl sulfate

polyacrylamide gel electrophoresis) .

1. Since the samples have been denatured in gel loading

buffer containing SDS detergent, the protein is uniformly

negatively charged and will now migrate in an electric

field through the gel and towards the positive electrode .

SDS-PAGE ( sodium dodecylsulphate-polyacrylamide gel electrophoresis)

The purpose of this method is to separateproteins according to their size, and noother physical feature.

Stacking Gel

Transfer of the proteins fractionated by SDS-PAGE

to a solid support membrane (Western blotting)

can be accomplished by electroblotting

In this procedure, asandwich of gel and solidsupport membrane(Nitrocellulose or PVDF) iscompressed in a cassetteand immersed in bufferbetween two parallelelectrodes.

A current is passed at rightangles to the gel, whichcauses the separatedproteins to electrophoreseout of the gel and onto thesolid support membrane

The membrane supports used in Western blotting have ahigh affinity for proteins. Therefore, after the transfer of theproteins from the gel, it is important to block the remainingsurface of the membrane to prevent nonspecific bindingof the detection antibodies during subsequent steps.

A variety of blocking buffers ranging from milk or normalserum to highly purified proteins have been used to blockfree sites on a membrane. The blocking buffer shouldimprove the sensitivity of the assay by reducingbackground interference and improving the signal tonoise ratio. No single blocking agent is ideal for everyoccasion since each antibody-antigen pair has uniquecharacteristics. For true optimization, empirical testing ofblocking buffers is essential.

The balance of SDS in the transfer buffer,protein size, and gel percentage are themain factors that affect transfer efficiency.

About the current and transfer time forwestern blot, it's critical to choose theappropriate current and transfer time for asuccessful western blotting.

Too low current or/and transfer time willlead to incomplete transfer; if the currentor/and transfer time is too high, on thecontrary, the proteins may migratethrough the membrane too fast withoutbeing absorbed.

One of the critical features of any successful Western blot is the highly

specific interaction between an antibody and an antigen. The antigen,

usually a protein or peptide, is the target of the antibody. The precise point

of interaction is between a small region of the antigen, an epitope,

and the recognition sites found on the arms of the antibody molecule.

1.The confirmatory HIV test

2.Western blot is also used as the definitive test for

Bovine spongiform encephalopathy (BSE(

3.Some forms of Lyme disease testing employ Western

blotting .