Western Blotting

Introduction

• Western blotting: an immunodetection technique used by proteomic scientists to detect and quantify specific proteins in complex biological samples.
• First, proteins are extracted from a sample of cells or tissue.
• Extracted proteins are loaded into a sieving gel matrix and separated according to size using an electric current, that is, by electrophoresis.
• Proteins separated by electrophoresis are then transferred or "blotted" from the gel onto a paper-like membrane.
• A specific antibody, engineered to bind only to the protein of interest, is added to the membrane.
  • This antibody is attached to a compound that causes a colored reaction, enabling scientists to detect and quantify a single protein of interest from hundreds of other proteins in a sample with high accuracy.
• Antibodies: key proteins found in all animal immune systems that detect foreign invaders such as viruses, bacteria, and allergens and tag them for destruction.
• Western blotting can categorically identify a specific protein among hundreds or thousands of other proteins within biological samples.
• Western blotting is based on two distinguishing features of proteins: molecular mass and antibody binding specificity.

Steps

• Proteins must first be transferred or "blotted" from within the gel onto the surface of a membrane.
• A membrane is more stable and longer lasting than a gel and proteins bound to the surface of a membrane are more accessible to antibodies.
• Proteins, still negatively charged from the SDS, migrate out of the gel and bind to the surface of the membrane, creating a mirror image of proteins separated in the original gel.
• The blot must be incubated in a protein-rich solution such as one derived from powdered milk protein.
  • Incubating the blot with milk protein effectively coats the entire surface area of the membrane where no proteins have been blotted and blocks nonspecific protein binding sites.
• Once proteins are transferred to the nitrocellulose membrane, the next step is to probe the blot with an antibody that has been specifically engineered to detect the protein of interest.
• Next the blot is incubated with an antibody engineered to bind only to the proteins (the primary antibody).
• Following a quick rinse, the membrane is incubated with an enzyme-linked secondary antibody that has been engineered to bind specifically to the primary antibody.
• Finally, a colorless colorimetric enzyme substrate is added to the membrane in solution.
• The enzyme that is linked to the secondary antibody oxidizes the colorimetric substrate into an insoluble colored precipitate, leaving a visible deposit on the membrane at the precise location of the blotted myosin light chain proteins.

Western Blot Reagents and Equipment

• Mini Tetra-Blot apparatus: the Mini Tetra-Blot is specifically designed to pass electric current horizontally through the gel, forcing the negatively charged proteins to migrate out of the gel onto the nitrocellulose membrane.
• Nitrocellulose membranes: nitrocellulose acts as a solid support for proteins bound to its positively charged surface.
  • These durable membranes can undergo multiple wash and incubation steps, and provide a white background on which to visualize the color development at the site of the protein of interest only.
• Blotting paper (or Filter paper): used to support the gel and nitrocellulose and to protect them from the fiber pads during assembly and electrophoresis.
  • The blotting paper also facilitates a uniform flow of buffer and current through the gel.
• Fiber pads: press the gel and nitrocellulose together tightly and uniformly, eliminate air bubbles, and allow efficient transfer of proteins out of the gel and onto the membrane.
• Blotting buffer: contains tris to maintain pH, glycine ions to transmit current, and ethanol to facilitate protein binding to the nitrocellulose.
• Blocker: prevents the primary antibody from randomly adhering to the membrane and obscure or weaken the specific antibody signal.

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