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Streptavidin is widely used in Western blotting and immunoassays conjugated to some reporter molecule, such as horseradish peroxidase. Streptavidin has also been used in the developing field of Nanobiotechnology, the use of biological molecules such as proteins or lipids to create nanoscale devices/structures.
Horseradish peroxidase is a 44,173.9-dalton glycoprotein with six lysine residues which can be conjugated to a labeled molecule. It produces a coloured, fluorimetric [6] or luminescent derivative of the labeled molecule when incubated with a proper substrate, allowing it to be detected and quantified.
Streptavidin is a tetrameric protein expressed in Streptomyces avidinii. Because of Streptavidin's high affinity for vitamin H , Streptavidin is commonly used in the fields of molecular biology and biotechnology. The Strep-tag was originally selected from a genetic library to specifically bind to a proteolytically truncated "core" version of ...
The Streptavidin-Binding Peptide (SBP)-Tag is a 38-amino acid sequence that may be engineered into recombinant proteins. Recombinant proteins containing the SBP-Tag bind to streptavidin and this property may be utilized in specific purification, detection or immobilization strategies.
Then, HRP-conjugated streptavidin is used for detection. [6] [11] HRP then converts diaminobenzidine (DAB) into an insoluble brown product, which can be detected in a bright-field microscope under 40- to 60-fold magnification. [11] [13] Staining lightly with hematoxylin can be used as a counterstain to make the product more visible. [5]
If streptavidin-HRP (streptavidin and horseradish peroxidase conjugate) is used, then using TMB (tetramethylbenzidine) [6] as a substrate will produce better results. [ 7 ] Analysis : The spots that are formed can then be read on an automated ELISpot reader, or counted under a dissection microscope, and further used to calculate the frequency ...
Extent of biotinylation can also be measured by streptavidin gel-shift, since streptavidin remains bound to biotin during agarose gel electrophoresis or polyacrylamide gel electrophoresis. The proportion of target biotinylated can be measured via the change in band intensity of the target with or without excess streptavidin, seen quickly and ...
Western blot workflow. The western blot (sometimes called the protein immunoblot), or western blotting, is a widely used analytical technique in molecular biology and immunogenetics to detect specific proteins in a sample of tissue homogenate or extract. [1]