Western Blot

Western blot ( Western blot ) and immunoblot ( Data Sheet immunoblot ) denotes the transmission (English blotting) of proteins on a carrier membrane, which can then be detected by different reactions. The transfer can be done in different ways: by diffusion, capillary action or electrophoresis. Applies the Western blot in biochemical and medical research and in diagnostics.

The Western blot method was originally developed at Stanford University in 1979 in the laboratory of Robert Nowinski at Fred Hutchinson Cancer Research Center in Seattle by W. Neal Burnette and independently in the laboratory of George R. Stark. In the same year Harry Towbin and colleagues were able to switch the method as in the simpler Southern blotting to nitrocellulose, which today is the simpler and preferred method.

The name of the blot method (" Western blot " ) comes from the English for blob blot or stain and Engl. blotting paper for blotting paper, in which even an identical print of the original image is created. This was first introduced in 1981 by Neal Burnette as an allusion ( the publication appeared two years late because the magazine had originally rejected). Edwin Southern is considered the inventor of the blotting technique. In 1975, he developed a method for the separation of DNA fragments and subsequent hybridization, which he described as a Southern blot. The corresponding separation of RNA fragments was designated according to its name as Northern blot. Therefore he called the protein blotting with SDS Western blot.

A Eastern Blot per se does not exist. However, the term " Eastern blot " is taken for several methods in claim, eg for an electrophoretic separation, and transfer of the proteins on the membranes with a cationic detergent (e.g. CTAB or 16 -BAC ), in which the proteins in the opposite direction ( towards the cathode ) hike. The term Eastern blot was used for the blotting of lipids in membranes, transfer of native proteins from non-denaturing gels, or dropping (English blotting) of molecules.

Principle

Before the actual Western blot, a protein mixture by means of gel electrophoresis in a carrier matrix (SDS-PAGE, native PAGE, isoelectric focusing, 2-D gel electrophoresis, etc.) separated according to their size, charge or other characteristics. Here, the proteins to be analyzed are first separated by gel electrophoresis ( usually a polyacrylamide gel with an optimum acrylamide concentration) in the protein bands.

Protein transfer

In Western blot a perpendicular to the polyacrylamide gel directed electric field is applied ( electron transfer ), so that the proteins migrate in the direction of the anode. If there is no time pressure, the transfer can alternatively be effected by capillary action in the direction of a dry stack of a hydrophilic absorbent material ( capillary ). During transfer, the proteins migrate out of the gel to a membrane, such as nitrocellulose, nylon, glass fiber or most of polyvinylidene fluoride ( PVDF). With nylon or PVDF proteins due to hydrophobic and polar interactions adhere to the membrane surface, while the adsorption at nitrocellulose or glass fibers is effected by means of ionic and polar interactions. When transferring the pattern of electrophoretic separation is maintained. The proteins are now but for other methods available (for example, binding of an immunoconjugate ). After this process, the accreted to the SDS proteins can be washed. Therefore, the proteins can renature and partially assume its secondary and tertiary structure again, due to the spatial separation of the different subunits of a protein, the quaternary structure and yet can be not restored. For determining the number and size of the subunits of a protein, however, prior to SDS -PAGE is a covalent cross-linking of the subunits can be performed, which is one boiling in sample buffer for SDS -PAGE and after immunostaining can reveal the structure of a protein complex. For electrophoretic transfer of two different systems are used: the tank blotting system and the semi-dry blotting system, which differ in construction and used buffer volumes and systems.

Protein Detection

The temporary dyeing of all proteins on the blot membrane allows a review of the protein transfer, and an estimation of amount of protein in the different lanes of the gel prior to immunodetection. The totality of the membrane-bound proteins can be visualized on certain dyes ( loading control ). Examples are Ponceau S, Colloidal gold, or amido black ink. Other dyes are able to select post-translational modifications such as phosphorylated proteins. The dyes commonly used in the SDS-PAGE as Coomassie staining or silver staining only allow a low renaturation of the proteins during the discoloration from immune detection and also discolor incomplete. Fluorescent dyes have in two-dimensional applications, a larger linear dynamic range, and allow more accurate quantification, such as total protein staining with trichloroethanol or Epicocconon. Following immunodetection as described in the next section takes place. Depending on the experimental set-up selective individual proteins by other methods can be made visible, for example, radioactively labeled antibodies or other proteins that are radioactively labeled by the incorporation of isotopes in the protein synthesis or by subsequent phosphorylation with enzymes by reacting a corresponding substrate.

Immunodetection of individual proteins

The protein bands are generally identified on the membrane using specific antibodies. -Specific antibody ( monoclonal), or a mixture of specific antibodies ( polyclonal) to bind to the appropriate protein band on the membrane. Non-specifically bound antibodies are removed due to washing steps with buffers containing detergents. It makes use of the affinity of binding between antigen and antibody advantage: An antigen-specific primary antibody binds to " be " epitope on the spatially separated from the other proteins antigen with a characteristic molecular weight. Because the restoration is not complete, can with the use of monoclonal antibodies that a discontinuous epitope ( synonym: conformational epitope ) recognized by the protein problems. With antibodies that a continuous epitope ( synonym: sequence epitope ) can detect a restoration is not required.

Detection is carried out mostly with a immunoconjugate. In the Fc region of the primary antibody, a secondary antibody as the antibody conjugate ( immunoconjugate ) binds to a reporter enzyme, the detection is performed on the other washes. The use of a secondary antibody conjugate instead of a primary antibody conjugate allows its modular use in various primary antibodies with concomitant cost savings since the coupling of each of the primary antibody is omitted with a reporter enzyme. It comes through the secondary antibody for signal amplification, since the polyclonal directed against multiple epitopes on the Fc fragment of a species secondary antibody can bind to multiple sites in the Fc region of all the primary of a kind and there grouped many reporter enzymes. Reporter enzyme - antibody conjugates ( immunoconjugates ) are commercially available.

Depending on the specific question, the immunoblot - as described below - the antigen or - such as the HIV test - also the primary to be the search object. Also Antibodies are proteins and therefore their antigenic properties can be next to the Western blotting, for example, by immunoblotting, ELISA and ELISPOT make visible.

Expiration

A typical Western blot may look like this:

• After the transfer (SDS- PAGE and blotting ) of proteins on the membrane must first be blocked, the free non-specific protein binding sites on the membrane, as this would cause the antibodies attach to these binding sites and make specific detection of antigens impossible. The blocking of free binding sites occurs with no apparent for antibody protein or chemical polymer. This is solution of defatted milk powder, bovine serum albumin (BSA, bovine serum albumin ), gelatin and other proteins, or even solutions of polyvinylpyrrolidone with a mild detergent suitable. An undyed size markers ( synonym: Komigrationsstandard ), in addition to the proteins to be examined used should be a reversible staining of all proteins to the membrane with, for example, Ponceau S first. The then visible marker bands can be obtained with a mechanical pressure on the diaphragm and be also used even after the color reaction, for protein identification. In prestained size markers reversible protein staining is omitted.
• The membrane is then treated with a dilute solution of antibodies, wherein the antibodies are specifically directed against a protein or against a number of proteins on the membrane.
• Some washing steps with a detergent solution to remove weakly adherent, non-specifically bound antibody from the membrane.
• A second antibody solution ( the secondary antibody ) is added to the membrane, the antibodies are directed specifically against particular regions of the first antibody and to bind (typically the Fc region of the antibody ) in these areas.
• After further washing steps now takes place depending on the detection method, the visual. For enzyme - immunoconjugates a color or chemiluminescent reaction is catalyzed by the enzyme.

Immunoblot vs. ( EL) ISA

Both are methods for the detection of antigens ( proteins ) are using labeled antibodies: Both are so ISAs ( immunosorbent assay), methods from the field of proteomics.

Immunoblotting extends the ELISA in a sense, the dimension of the electrophoretic separation at the expense of a selective enrichment of the coating antigens by antibodies. This lack of enhancement can be achieved by an additional protein purification or immunoprecipitation. By gel electrophoresis, and the fixation on a solid medium ( the blot membrane ) are the antibodies to various defined locations different antigens isolated from the " Selection": A single immunoblot For example, a serum by a variety aufgeblotteter antigens on precisely this variety of associated antibodies check, however, almost exclusively continuous epitopes are detected (synonym Sequenzepitope ) due to denaturation during sample preparation for SDS -PAGE. The spatial separation can also several components against a serum containing antibodies that are detected in parallel, and selective. This effect can be achieved by sera from immunized or convalescent (polyclonal ) or by mixtures of monoclonal antibodies.

Applications

In the field of protein biochemistry of the Western blot is used to detect specific proteins and protein modifications, such as phosphorylation. It can also be performed a semi-quantitative analysis of (Sample A contains more protein X as Sample B), with the application of a dilution series of a known concentration of protein, the estimation of the amount on the blot may be compared in more detail.

In the field of medicine, the Western blotting is used to detect diagnostically relevant proteins such as antibodies in the serum, which may be typical of the presence of certain infectious diseases. By means of the Western blot can be prepared test strip to detect antibodies against specific viruses in the serum, for example. This method also helps in medical research in the search for disease-related proteins such as the BSE agent or HIV. Proteins such as the ERK, which occur frequently in tumors can be quantified via Western blot and degenerate cells are recognized by this. This can be determined, for example, to what extent certain medications regulative effect on the increased expression of these proteins in the cell and thus have a regulatory effect on the further growth of tumor cells.