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Western blot is also known as immunoblot protein because antibodies are mainly used to detect the antigen. It is a widely accepted analytical technique used to detect specific proteins in a specific sample.
Western blotting is commonly used in protein isolation and identification studies.
A Western blot approach is used to find protein from a raw mixture. You can find western blot analysis services at https://www.bosterbio.com/services/c-elegans-zebrafish-gene-editing-services.
First, the proteins are separated into strips by gel electrophoresis. All proteins are then transferred to the membrane, where the target protein band is identified by the primary antibody.
The target protein is more specific. Such primary antibodies on the membrane are detected by secondary antibodies that are enzymatically or fluorescently labelled.
All of these techniques use antibodies to identify proteins in tissues and cells by immunostaining and enzyme-linked immunosorbent assay (ELISA).
The immunometric test, also known as a sandwich ELISA, uses two precise antigen antibodies to capture the antigen for detection.
WESTERN BLOT WORKFLOW
- Transfer: Proteins are transferred through a gel on a membrane made of nitrocellulose (NC) or polyvinylidene difluoride (PVDF). There is no chemical formula or pre-activation due to hydrophobic interactions.
- Blocking: In Western blotting, it is important to block unreacted areas of the membrane. This reduces the binding amount of non-specific proteins by following the steps in testing using an inert protein or nonionic detergent.
- Primary incubation of antibodies: blocking, primary immunodeficiency antibodies specific to the target protein growing in the membrane. Additionally, primary antibodies are combined to target proteins to the membrane.
- Secondary Antibody Incubation: Flushing the membrane to remove the unbound primary antibody will expose the membrane to the final enzyme-conjugated antibody. In addition, secondary antibodies bind to primary antibodies, which are cross-reactive with target proteins.