Western Blotting2

8/9/2019 Western Blotting2

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WESTERN BLOTTINGPresentation by: Mariela T.Alcaparas
http://en.wikipedia.org/wiki/File:Immunoblot.png


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WESTERN BLOT

The western blot or proteinimmunoblotis an analytical technique used to detectspecific proteins in a given sample of tissuehomogenate or extract.

It uses gel electrophoresis to separate nativeor denatured proteins by the length of thepolypeptide (denaturing conditions) or by the3-D structure of the protein (native/ non-denaturing conditions).

The proteins are then transferred to amembrane (typically nitrocellulose or PVDF),where they are detected using antibodiesspecific to the target protein.


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The method originated from the laboratory ofGeorge Stark at Stanford.

The name western blotwas given to thetechnique by W. Neal Burnette and is a playon the name Southern blot, a technique forDNA detection developed earlier by Edwin

Southern. Detection of RNA is termednorthern blotting and the detection of post-translational modification of protein istermed eastern blotting.


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STEPS IN A WESTERN BLOT

Tissue preparation Samples may be taken from whole tissue or from cell

culture.

In most cases, solid tissues are first broken down

mechanically using a blender (for larger samplevolumes), using a homogenizer (smaller volumes).

However, it should be noted that bacteria, virus orenvironmental samples can be the source of proteinand thus Western blotting is not restricted to cellular

studies only. Protease and phosphatase inhibitors are often added

to prevent the digestion of the sample by its ownenzymes. Tissue preparation is often done at coldtemperatures to avoid protein denaturing.


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A combination of biochemical and mechanical techniques including various types of filtration and centrifugation can beused to separate different cell compartments and organelles.
http://en.wikipedia.org/wiki/File:SDS-PAGE_sample.png


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Gel electrophoresisThe proteins of the sample are separated using gel

electrophoresis.

Separation of proteins may be by isoelectric point (pI),molecular weight, electric charge, or a combination ofthese factors.

The nature of the separation depends on the treatmentof the sample and the nature of the gel. This is a veryuseful way to determine a protein.

By far the most common type of gel electrophoresisemploys polyacrylamide gels and buffers loaded withsodium dodecyl sulfate (SDS).

SDS-PAGE (SDS polyacrylamide gel electrophoresis)maintains polypeptides in a denatured state once theyhave been treated with strong reducing agents to remove

secondary and tertiary structure (e.g. disulfide bonds andthus allows separation of proteins by their molecularweight.


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The concentration of acrylamide determines theresolution of the gel - the greater the acrylamideconcentration the better the resolution of lowermolecular weight proteins. The lower theacrylamide concentration the better theresolution of higher molecular weight proteins.Proteins travel only in one dimension along thegel for most blots.


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Samples are loaded into wells in the gel. One lane is usuallyreserved for a markeror ladder, a commercially available mixture ofproteins having defined molecular weights, typically stained so as toform visible, coloured bands. When voltage is applied along the gel,proteins migrate into it at different speeds. These different rates ofadvancement (different electrophoretic mobilities) separate intobands within each lane.


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Transfer In order to make the proteins accessible to antibody

detection, they are moved from within the gel onto amembrane made ofnitrocellulose or polyvinylidenedifluoride (PVDF).

The membrane is placed on top of the gel, and a stackof filter papers placed on top of that. The entire stack isplaced in a buffer solution which moves up the paperby capillary action, bringing the proteins with it.

Another method for transferring the proteins is calledelectroblotting and uses an electric current to pullproteins from the gel into the PVDF or nitrocellulosemembrane.

Protein binding is based upon hydrophobic interactions,as well as charged interactions between the membraneand protein. Nitrocellulose membranes are cheaperthan PVDF, but are far more fragile and do not stand upwell to repeated probings.


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Blocking

Blocking of non-specific binding is achieved by

placing the membrane in a dilute solution ofprotein - typically Bovine serum albumin (BSA)or non-fat dry milk (both are inexpensive), witha minute percentage of detergent such asTween 20.

The protein in the dilute solution attaches tothe membrane in all places where the targetproteins have not attached. Thus, when theantibody is added, there is no room on themembrane for it to attach other than on the

binding sites of the specific target protein. Thisreduces "noise" in the final product of theWestern blot, leading to clearer results, andeliminates false positives.


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Detection

During the detection process the membrane is"probed" for the protein of interest with a modifiedantibody which is linked to a reporter enzyme, which

when exposed to an appropriate substrate drives acolourimetric reaction and produces a colour. For avariety of reasons, this traditionally takes place in atwo-step process, although there are now one-stepdetection methods available for certain applications.


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Analysis After the unbound probes are washed away, the

Western blot is ready for detection of the probesthat are labeled and bound to the protein ofinterest. In practical terms, not all Westerns revealprotein only at one band in a membrane.

Size approximations are taken by comparing thestained bands to that of the marker or ladderloaded during electrophoresis. The process isrepeated for a structural protein, such as actin ortubulin, that should not change between samples.

The amount of target protein is indexed to thestructural protein to control between groups. This

practice ensures correction for the amount of totalprotein on the membrane in case of errors orincomplete transfers.

Western blot using radioactive detection system


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Colorimetric detectionThe colorimetric detection method depends on incubation

of the Western blot with a substrate that reacts with thereporter enzyme that is bound to the secondary antibody.

This converts the soluble dye into an insoluble form of adifferent color that precipitates next to the enzyme andthereby stains the membrane.

Development of the blot is then stopped by washing awaythe soluble dye. Protein levels are evaluated through

densitometry (how intense the stain is) orspectrophotometry.


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Chemiluminescent detection Chemiluminescent detection methods depend on

incubation of the Western blot with a substrate that will

luminesce when exposed to the reporter on the secondaryantibody.

The light is then detected by photographic film, and morerecently by CCD cameras which capture a digital image ofthe western blot.

The image is analysed by densitometry, which evaluatesthe relative amount of protein staining and quantifies theresults in terms of optical density. Newer software allowsfurther data analysis such as molecular weight analysis ifappropriate standards are used.


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Radioactive detectionRadioactive labels do not require enzyme

substrates, but rather allow the placement ofmedical X-ray film directly against the westernblot which develops as it is exposed to the labeland creates dark regions which correspond to theprotein bands of interest .

The importance of radioactive detectionsmethods is declining because it is veryexpensive, health and safety risks are high andECL provides a useful alternative.


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Secondary probingOne major difference between nitrocellulose and PVDF

membranes relates to the ability of each to support"stripping" antibodies off and reusing the membrane forsubsequent antibody probes.

While there are well-established protocols available forstripping nitrocellulose membranes, the sturdier PVDFallows for easier stripping, and for more reuse before

background noise limits experiments. Another difference is that, unlike nitrocellulose, PVDF

must be soaked in 95% ethanol, isopropanol ormethanol before use. PVDF membranes also tend to bethicker and more resistant to damage during use.


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Medical diagnostic applications

The confirmatory HIV test employs a Western blot to detectanti-HIV antibody in a human serum sample. Proteins fromknown HIV-infected cells are separated and blotted on a

membrane as above. Then, the serum to be tested is appliedin the primary antibody incubation step; free antibody iswashed away, and a secondary anti-human antibody linked toan enzyme signal is added. The stained bands then indicatethe proteins to which the patient's serum contains antibody.

A Western blot is also used as the definitive test for Bovinspongiform encephalopathy (BSE, commonly referred to as'mad cow disease').

Some forms of Lyme disease testing employ Western blotting.

Western blot can also be used as a confirmatory test forHepatitis B infection.

In veterinary medicine, Western blot is sometimes used toconfirm FIV+ status in cats.


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