Western blot analysis (immunoblotting) is high sensitive temporary analytical method used for the determination of specific proteins with antibodies. The method is based on a combination of gel electrophoresis and immunochemical reaction named “antibody/antigene (target protein).” The high degree of resolution is achieved by electrophoretic separation of proteins and specificity of mono- or polyclonal antibodies. The optimal conditions of Western blot allow detecting less than 1 ng proteins.
Western blotting stages:
- Separation of proteins by SDS-electrophoresis. The most common method for protein separation is electrophoresis with sodium dodecyl sulfate (SDS.). In this case all proteins possess only negative charge and they can be split, depending only on their molecular weights. Denatured proteins are loaded to a specially shaped “track” and migrate into the electric field through the acrylamide gel, while smaller proteins move faster. One of the “tracks” is used for molecular weight markers (protein with known weights). Differences in the rates of distribution (electrophoretic mobility) lead to the separation of proteins in the band.
- Protein transfer to membrane. Electroblotting transfer of proteins from gel to membrane (nitrocellulose or polyvinylidene fluoride (PVDF)) takes place under an electric current. Proteins are transferred from the gel to membrane well preserving their location. In this process, the proteins are in a thin surface layer of the membrane and available for further binding with antibodies. The binding of proteins is based on both hydrophobic and electrostatic interactions at the membrane and between the protein. Thus, after electrotransfer on nitrocellulose we obtain replica of the gel with proteins located in the same way as in a polyacrylamide gel.
- Blocking of non-specific binding antibodies with membrane is achieved by incubation of the membrane with a dilute solution of bovine serum albumin or skim milk powder. Proteins from a dilute solution bind with membrane in those places where bands are not located. As a result, when antibodies are added they can bind only specific binding sites of the target proteins. Blocking can achieve a clean background and excludes obtaining false-positive results.
- Binding target protein with antibodies. After blocking, the membrane is 3-fold washed with buffer and sequentially incubated with various antibodies by ‘sandwich’: firstly the proteins bind with the primary (mono- or polyclonal) antibodies. They, in turn, communicate with the secondary antibodies, conjugated with enzymes (horseradish peroxidase or alkaline phosphatase).
- Detection. Visualization of the protein under study is achieved by appropriate biochemical reaction to form a product which is determined by colorimetric, chemiluminescent, fluorescent detection methods. The amount of protein can be estimated by densitometry.
It is planned to estimate a certain amount of light-harvesting complex of photosystem 2 protein – Lhcb2 from Arabidopsis thaliana thylakoids.
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