Western blot

A buvardage of western or western blot (or immunoblot ) is a method of Protéomique, having recourse to the Molecular biology, the Biochimie and the Immunogénétique, to detect a specific Protéine in a given sample of extract or tissue Homogénat. The technique uses the electrophoresis on polyacrylamide gel to separate proteins, beforehand denatured, according to their mass. The proteins are then transferred since freezing on a membrane (typically in Nitrocellulose), where they are exposed to a specific Anticorps of protein of interest. It is possible thanks to this technique to detect the presence of a protein in a fabric, to evaluate its size, its concentration, the variations of this concentration, to carry out comparisons of concentrations between various groups, etc Of other techniques using the antibodies allow the detection of protein in the cells after fixing (Immunocytochimie) and in the fabrics (Immunohistochimie).

The method was developed in the laboratory of George Stark in Stanford. The name of the western blot , given to the technique by W. Neal Burnette (Analytical Biochemistry, 112:195 - 203, 1981) , is a pun starting from the technique of the Southern blot or buvardage of Southern, technique of detection of DNA named according to its inventor, Edwin Southern and not according to the cardinal point. The detection of ARN is called Northern blot or buvardage of northern. All these techniques derive their name from the stage of transfer on membrane , compared with a print on Buvard ( blot in English)

Various stages of the buvardage of western

Preparation of the samples

Typically, the samples are taken of a fabric or a cellular culture. They quickly are cooled, even cooled (in lower part of 0°C). They are homogenized by Sonication (use of ultrasounds to break the membranes), forced mechanical or simply lysed by use of plugs with high salt concentrations. It results a homogenate from it from all the cellular compartments, being able to be used such as it is or to be subjected at several stages of differential centrifugations in order to isolate the fractions ic Cytosol, nuclear and membrane. The sample is then treated in order to collect a constant protein rate starting from each different sample. That implies a proportioning of proteins by the method of the Biuret or the blue of Coomassie (Méthode of Bradford).

The samples are then boiled from 1 to 5 minutes in a Buffer solution (for example the plug of Laemmli), containing a substance plug, generally Tris, a dye, a component sulfhydryl (typically of the Beta-mercaptoéthanol or Dithiothréitol or more simply DTT), a anion Détergent lipophilic (sodium dodécyl sulfate or SDS) and Glycérol to increase the surface stress.
L' boiling denatures the proteins by breaking the intramolecular connections weak, which results in to unroll them completely. The SDS then gets to them an environment rich in negative charges in order to them solvater and to prevent precipitation, and composing it sulhydryl prevents the reformation of Pont disulfide. The glycerol increases the density of the sample compared to the plug in the upper part of the reserve of freezing, facilitating the setting of the samples which will go down more easily at the bottom from the compartments from freezing.

Electrophoresis on freezing

The proteins of the sample are separate according to their size by electrophoresis on freezing, the composition whose varies according to the laboratory, of the Molecular weight of proteins of interest, and the plugs available. the polyacrylamide gels are most frequent. The proteins crossing freezing only in one dimension (the top downwards), the samples are charged one beside the other in " puits" formed in freezing. The proteins are separated by mass in " bandes" in each " couloir" formed under the wells. A corridor is reserved for a " marqueur" or " scale standard" , a protein mixture having of the definite molecular weight available in the trade.

It is also possible to employ a freezing 2D which, starting from one only sample, makes it possible to make migrate proteins in two dimensions. The proteins are then separated by their isoelectric point (i.e. pH to which them clear load is neutral) in the first dimension, and according to their weight in the second.

The composition of a plug of electrophoresis ( Running buffer ) for western blot is of 1 volume of plug TGS (Tris - glycine - SDS) concentrated 10 times in 9 volumes of distilled water.

Transfer on membrane

In order to make proteins accessible to detection by antibody, they are transferred since freezing on a membrane from Nitrocellulose or PVDF. The membrane is placed face-to-face discussion with freezing, and an electric current is applied to the large plates to one on the two sides. The proteins charged migrate since freezing towards the membrane by preserving the relative organization which they had in freezing. It results from this transfer that the proteins are exposed on a mean surface, which facilitates the later stages of detection. As well nitrocellulose membranes as PVDF are " collantes" , binding proteins in a not-specific way (i.e. they bind all the proteins present in the sample in the same way). The fixing of proteins to the membrane is made thanks to hydrophobic Interactions and ic Ion between the membrane and proteins. Although the nitrocellulose membranes are less expensive than those in PVDF, they are less solid and cannot be employed several times. With the difference of those out of nitrocellulose, the membranes of PVDF must be soaked with Méthanol or Isopropanol to 100% before their use.

The composition for one liter of a standard plug of transfer is of:

700 ml of distilled water
100 ml of plug TG (Sorting - glycine) concentrated 10 times
200 ml of Methanol
4 ml of SDS

Blocking

The membrane having been selected for its properties of not-specific connection, as so much the protein-target which the antibodies are proteins, precautions must be taken to minimize the interactions between membrane and antibody. The blocking of the nonspecific sites of interactions between the membrane and the antibodies is carried out while plunging the membrane in a diluted protein solution - generally of the Albumine of bovine serum or ASB (English BSA) or milk without fat contents (diluted milk with 5% - 5 G for 100 ml) - in the presence of detergent, typically of Tween® 20), during one hour with room temperature.
Les proteins in the diluted solution binds to the membrane in all the sites not-occupied by the protein-target. This way, when the antibodies are applied at the time of the following stage, they cannot (ideally) stick more to the membrane but on the sites of connection of the protein-target, which reduces the " background noise " in the finished product of the buvardage of western, gives clearer results and eliminates the false-positives.

Detection

During the penetration, the membrane is " déchirée" for protein of interest with antibodies, then dependant on an enzyme emitting a photometric signal or colorimetric. For several reasons, this occurs classically in two stages, although methods in a stage are available for certain applications.

Method in two stages

Primary education antibody

The antibodies are generated by inoculation with the animal (generally a rabbit or a Chèvre) or exposure of a culture of immunizing cells to protein of interest in its entirety or only on one of its fractions (épitope). However, the proteins having been denatured during the electrophoresis on freezing, it is necessary to use antibodies which specifically recognize denatured protein, and not the protein native.
La normal immunizing response in this case is exploited in order to generate antibodies which are collected and used like tools for detection having at the same time a good sensitivity and a good specificity, binding directly to protein - from where their name of antibody " primaire". Some monoclonal antibodies, much more difficult to realize and whose affinity is much higher can also be used in buvardage of western.

After blocking, a diluted solution of primary education antibody (generally ranging between 0.5 and 5 microphone grams/ml) is incubated with the membrane under moderate agitation. The solution is composed typically of a saline plug near to neutral pH (generally, a small quantity of Sodium chloride), of a small percentage of detergent, and sometimes of proteins (ASB or milk 5%) diluted. The solution of antibody and the membrane can be sealed in a plastic bag and incubated together for a duration going from 30 minutes to one night. It can also be incubated at various temperatures, the higher temperatures being associated with more specific connections.

Secondary antibody

After rinsing of the membrane in order to remove the nondependant primary education antibodies, this one is exposed to another antibody, directed against a species-specific portion of the primary education antibody. This antibody is known like antibody " secondaire" , and tends to being referred, because of its properties of targeting, like " anti-mouse, " " anti-goat, " " anti-lapin" , etc the antibodies are of animal source (but generally, of different species), or of cultures of Hybridome S of animal origin; an antibody anti-mouse will bind to practically any primary education antibody of murine origin, which makes it possible to realize savings by letting the laboratory share only one source of secondary antibody, and allows more reproducible results. The secondary antibody is generally dependant on the Biotine or a Enzyme which allows the visual identification of protein studied on the membrane, like a alkaline Phosphatase or the Peroxydase of horseradish. This stage confers an advantage, in what several secondary antibodies will bind to a primary education antibody, allowing to improve the signal.

Most commonly, a secondary antibody related to the Peroxydase of Raifort (horseradish peroxidase) is used in conjunction with a luminescent agent, and the product of the reaction emits a Luminescence proportional to the protein concentration. A sensitive photographic film is placed against the membrane and, under the exposure of the light due to the reaction, creates an image of the antibodies related to the membrane. More often now, a camera with CCC is used in place of photographic films.
Comme for the procedures of ELISPOT and ELISA, the Enzyme can be provided with a molecule-substrate which will be converted by the enzyme to emit a coloured product of reaction, visible on the membrane.

A third possibility consists in using a radioactive marker rather than an enzyme coupled with the secondary antibody, for example by marking a protein binding the antibodies, such as protein has Staphylocoque with a radioactive Isotope of the Iode. However, the other surer, faster and less expensive methods being, this method fell more or less in disuse, but remains sometimes used in certain circumstances.

Method in a stage

When the technique appeared, the process of detection was carried out in two stages, because of relative facility of production of the primary education antibodies and secondaries in two distinct processes. That allows the researchers and suppliers a certain flexibility employment, and adds a stage of amplification of the signal to the process of detection. However, since the arrival of high-output protein analyzes and with margin of lower detection, i.e. detecting proteins very weak concentration has, it became interesting to develop systems of survey in a single stage which will allow a time-saver and a saving in raw material. These systems require an antibody of detection which can at the same time detect protein of interest and emit a detectable signal, which are often available for markers of known protein. The primary probe is incubated with the membrane with the manner of the primary education antibody in the process in two times, and is then ready for direct detection after a series of stages of rinsing.

Analyzes

After rinsing of the not-dependant secondary antibodies, the western blot is ready for the detection of the probes marked and been dependant on protein of interest. In practice, all the buvardages of western do not reveal the protein on a tape given of the membrane, the gel not being completely free from proteins after having sponged. An approximation on the size is carried out by comparing the bands marked with those of the markers of the range standard charged lasting the electrophoresis in a well with share. The process is repeated for a protein of structure, like the Actine or the Tubuline, whose concentration should not vary between the samples (internal control). The concentration of the protein-target is indexed with that of protein being used as internal control, in order to standardize the experiments. This practice allows the correction compared to the total protein rate on the membrane, in the event of error or of incomplete transfer.

Colorimetric detection

This method is tributary, during the incubation of the buvardage of western, the presence of a substrate which reacts to the release present on the secondary antibody (like the Peroxydase). This converts a soluble dye into an insoluble form, of different color, which precipitates beside the enzyme, thus dyeing the nitrocellulose membrane. The development of the buvardage of western is then stopped by rinsing of the soluble dye. The protein concentration is evaluated by Densitométrie, evaluation of the intensity of the band or by Spectrophotométrie.

Detection by chimiluminescence

This method requires, during the incubation of the buvardage of western, the presence of a substrate which emits light after exposure to the release present on the secondary antibody. The emitted light is used to impress a photographic film, or more recently, by cameras CCC which restore an digital image of the buvardage of western. The image is analyzed by densitometry, which evaluates the relative rate of marking of protein, and quantifies the results in optical term of Densité. Logiciel S allow a more thorough analysis of the data, as the analysis of the molecular weight if the suitable standards are used. This method called " Detection improved of the chimiluminescence " (" enhanced chemiluminescent" - ECL) is regarded as one of the most significant methods of detection for the analysis of the western blots.

Detection by radioactivity

Radioactive marking does not require an enzymatic substrate, but allows the use of films used in medicine for the imagery x-rays. the film is directly placed on the buvardage of western, which develops during its exposure to the marker and creates dark areas, which correspond to the bands of protein of interest (cf illustration). The importance of the methods of detection by radioactivity is declining, because of its cost and of surer techniques like the ECL.

Detection by fluorescence

The probe coupled with the antibody is excited by a monochromatic ray and the emission which results from it is then detected by a photosensor, for example a camera CCC equipped with the adapted filters of emission, which restores an digital image of the buvardage of western and allows a finer analysis such as the analysis of the molecular weight or quantitative of the buvardage of western. Fluorescence is regarded as level about equivalent to the Chimiluminescence for the analysis of the buvardages of western. It requires more expensive tools however.

Difference between Fluorescence and Chimiluminescence

Although of similar mechanism photophysic, chimiluminescence and fluorescence are not the same thing:

the Fluorescence refers to the excitation of a molecule since a stable condition towards a excited state, and its return at the basal state by emission of a radiation in the electromagnetic spectrum a given, and specific wavelength of the molecule.
the Chimiluminescence refers to the situation in which a molecule is formed in an excited state, as a product of a chemical reaction, and falls down towards a basal state with emission of a radiation in the electromagnetic spectrum a given wavelength.

Secondary survey

One of the greatest differences between the membranes out of nitrocellulose and those in PVDF is related to their capacity to support l'" arrachage" ( stripping ) of antibody and the re-use of the membranes for surveys by other antibodies. Although there exist protocols drawn up well for the re-use of the nitrocellulose membranes, the PVDF, thicker, makes it possible to carry out these operations in full safety and facilitated, and more later uses before being, a such Palimpseste, covered with " bruits" parasites. Another difference importance is that the PVDF, contrary to nitrocellulose, must be soaked in ethanol with 95% or isopropanol before use. The membranes in PVDF also tend to being thicker and more resistant to the physical damage related to their normal use.

Medical applications in diagnosis

the tests HIV of confirmation employ the method of the buvardage of western in order to detect an anti-HIV antibody in a sample of serum. Proteins of cells which one knows infected by HIV are separated and transferred on membrane as described above. The serum to be tested is applied. The stage of incubation in the primary education antibody; the free antibodies are eliminated by rinsing from the membrane, and an antibody directed against secondary human proteins associated with an enzyme or chromophoric is added. The marked bands indicate then the proteins against which the serum of the patient contains antibodies.
the buvardage of western is also used for the test of confirmation of ESB (known as “disease of the mad cow”).
Certaines forms of detection of the disease of Lyme uses the buvardage western.

References

Renart J, Reiser J, Stark GR. " Transfer off proteins from gel to diazobenzyloxymethyl-paper and detection with will antisera: method for studying antibody specificity and antigen structure" has; , Proc Natl Acad Sci U S At , 1979 Jul; 76 (7): 3116-20. PMID: 91164 abstract
Towbin H, Staehelin T, Gordon J. " Electrophoretic transfer off proteins from polyacrylamide gel to nitrocellulose sheets: procedure and nap applications" Proc Natl Acad Sci U S At , 1979 Sep; 76 (9): 4350-4. PMID: 388439 abstract
Burnette WN. " Western blotting": electrophoretic transfer off proteins from sodium dodecyl sulfate--polyacrylamide gel to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein has, Anal Biochem. , 1981 Apr; 112 (2): 195-203. PMID: 6266278 DOI: 10.1016/0003-2697 (81) 90281-5
Citation' S Classic: Burnette

See too

Southern blot (DNA), always used but of interest slightly in fall since PCR
Northern blot (ARN)
Far-western blot (interactions between proteins)

External bonds

Explanations and mechanisms Elisa and Western Blot with figures
Western Blot Protocol

Random links: