Western Blotting: Difference between revisions
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Protocol for Western Blotting: | Protocol for Western Blotting: | ||
#Prepare Tissue | #Prepare Tissue | ||
#[[Electrophoresis|Electrophoresis]] | #[[Electrophoresis|Electrophoresis]] | ||
#Transfer to polymer sheet | #Transfer to polymer sheet | ||
#Add [[Antibody|Antibodies]] | #Add [[Antibody|Antibodies]] | ||
#Identify | #Identify | ||
The protein is run on an [[ | The protein is run on an [[SDS polyacrylamide-gel electrophoresis|SDS-polyacrylamide gel]]. To enable the proteins to be detected the gel is transfered onto a polmer sheet, such as [[Nitrocellulose|nitrocellulose]], it is simply pressed against the gel transfering the protein to the sheet. This nitrocellulose sheet is then exposed to the [[Primary antibody|primary antibody]] that is specific to the protein that we want to identify. A different method to adding [[Secondary antibody|secondary antibody]] is the [[ELISA|ELISA]] method where an enzyme on the second antibody will produce a coloured product. | ||
Non-fat dried milk is often used in Western blotting as it binds to the membrane, preventing non-specific antibody binding and it reduces background signal. | |||
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Revision as of 15:13, 19 October 2015
Western Blotting (also known as immunoblotting) is a technique used in biomedicine to isolate and identify a protein of interest. Western Blotting involves the use of antibodies to bind to the protein (primary antibodies) and then to identify through bioluminescence (secondary antibodies). It is possible to not only identify the protein with the fluroescent label but we can also determine the quantity of the protein.
Protocol for Western Blotting:
- Prepare Tissue
- Electrophoresis
- Transfer to polymer sheet
- Add Antibodies
- Identify
The protein is run on an SDS-polyacrylamide gel. To enable the proteins to be detected the gel is transfered onto a polmer sheet, such as nitrocellulose, it is simply pressed against the gel transfering the protein to the sheet. This nitrocellulose sheet is then exposed to the primary antibody that is specific to the protein that we want to identify. A different method to adding secondary antibody is the ELISA method where an enzyme on the second antibody will produce a coloured product.
Non-fat dried milk is often used in Western blotting as it binds to the membrane, preventing non-specific antibody binding and it reduces background signal.