Western Blotting
WESTERN BLOTTING
(Written Report)
I. PRINCIPLE
Western blotting is a method for identifying a specific protein in a complex mixture and simultaneously determining its molecular weight. In Western blotting, proteins are electrophoresed into a gel, as the proteins migrate through the gel they are separated based upon size and charge. Characteristically, smaller proteins migrate through the gel faster than larger proteins. Sufficiently separated proteins in an SDS-PAGE can be transferred to a solid membrane (PVDF or Nitrocellulose) for WB analysis. For this procedure, an electric current is applied to the gel so that the separated proteins transfer through the gel onto the membrane. To detect the antigen blotted on the membrane, a primary antibody (serum) is added at an appropriate dilution and incubated with the membrane. Antibodies present in the serum bind to the protein(s). In order to detect the bound antibodies, anti-immunoglobulin antibodies coupled to an enzyme alkaline phosphatase or horseradish peroxidase are added. This anti-IgG enzyme is commonly called a "second antibody" or "conjugate". Finally, after excess second antibody is washed free of the blot, a substrate is added which will precipitate upon reaction with the conjugate resulting in a visible band where the primary antibody is bound to the protein.
The procedure can be broken down into a series of steps:
1. SDS Polyacrylamide Gel Electrophoresis (PAGE) by which proteins are separated according to their size. SDS binds strongly to most proteins, causes them to unfold to a random, rod-like chain, and makes them net negative in charge. No covalent bonds are broken in this process. Therefore, the amino acid composition and sequence remains the same. Proteins that loose their specific folding patterns and biological activity but their polypeptide chains remain intact, are called denatured. High concentrations of reducing agents, such as 2-mercaptoethanol, break disulfide bonds. This
(Written Report)
I. PRINCIPLE
Western blotting is a method for identifying a specific protein in a complex mixture and simultaneously determining its molecular weight. In Western blotting, proteins are electrophoresed into a gel, as the proteins migrate through the gel they are separated based upon size and charge. Characteristically, smaller proteins migrate through the gel faster than larger proteins. Sufficiently separated proteins in an SDS-PAGE can be transferred to a solid membrane (PVDF or Nitrocellulose) for WB analysis. For this procedure, an electric current is applied to the gel so that the separated proteins transfer through the gel onto the membrane. To detect the antigen blotted on the membrane, a primary antibody (serum) is added at an appropriate dilution and incubated with the membrane. Antibodies present in the serum bind to the protein(s). In order to detect the bound antibodies, anti-immunoglobulin antibodies coupled to an enzyme alkaline phosphatase or horseradish peroxidase are added. This anti-IgG enzyme is commonly called a "second antibody" or "conjugate". Finally, after excess second antibody is washed free of the blot, a substrate is added which will precipitate upon reaction with the conjugate resulting in a visible band where the primary antibody is bound to the protein.
The procedure can be broken down into a series of steps:
1. SDS Polyacrylamide Gel Electrophoresis (PAGE) by which proteins are separated according to their size. SDS binds strongly to most proteins, causes them to unfold to a random, rod-like chain, and makes them net negative in charge. No covalent bonds are broken in this process. Therefore, the amino acid composition and sequence remains the same. Proteins that loose their specific folding patterns and biological activity but their polypeptide chains remain intact, are called denatured. High concentrations of reducing agents, such as 2-mercaptoethanol, break disulfide bonds. This