Understanding Western Blot Technique

Western Blotting

• Western blotting, also known as immunoblotting, is a technique used to identify one specific protein in a sample containing many proteins.
• The technique involves separating proteins by gel electrophoresis, transferring them to a membrane, and then detecting the protein of interest using a specific antibody.

Steps of Western Blotting

• Step 1: Sample Preparation - involves lysing cells or tissue samples to release proteins, measuring protein concentration, and treating samples with agents to break higher-level structures.
• Step 2: Gel Electrophoresis - separates proteins based on their molecular weight using SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis).
• Step 3: Protein Transfer - transfers proteins from the gel to a membrane, such as nitrocellulose or polyvinylidene fluoride (PVDF), using an electric field.
• Step 4: Immunodetection - uses specific antibodies to detect the protein of interest on the membrane.

Preparing Samples for Electrophoresis

• Lysis buffer should be ice-cold and freshly-made, and protease inhibitor cocktail should be added to the buffer prior to lysis.
• The lysis buffer should be chosen based on the cellular localization of the protein of interest.
• Whole cell lysis buffer: NP-40 or RIPA; Cytoplasmic (soluble): Tris-HCl; Cytoplasmic (cytoskeletal bound): Tris-Triton; Membrane bound: NP-40 or RIPA; Nuclear: RIPA; Mitochondria: RIPA.

Reducing and Denaturing

• Reducing agents, such as β-mercaptoethanol or DTT, are used to break disulfide bonds and expose binding sites for the antibody.
• Denaturing agents, such as SDS, are used to break higher-level structures and coat proteins with negative charges at a uniform mass ratio.
• Note: Some antibodies only recognize non-reduced and/or non-denatured protein, so SDS and/or β-mercaptoethanol or DTT should be removed from buffers.

SDS-PAGE

• SDS-PAGE is a method to separate individual proteins from one another in an electric field according to electrophoretic mobility.
• Proteins are covered by SDS (negative charges) at a uniform mass ratio, so endogenous protein charges are negligible.
• Smaller proteins are less inhibited by the matrix and move more quickly.
• Gels can be made of different percentages of acrylamide to separate proteins of different sizes.

Protein Transfer

• The principle of protein transfer is similar to PAGE, where negatively charged proteins migrate toward the cathode in an electric field.
• A membrane is sandwiched between the gel and the cathode, and proteins adhere to the membrane in the same pattern as in the gel.

Immunodetection

• Antibodies are used to detect the protein of interest on the membrane.
• Detection methods include chemiluminescent substrates, chromogenic substrates, and fluorescent-conjugated secondary antibodies.
• Examples of antibodies used in Western blotting: Ab8226, Ab94303, Ab89345.

Learn about Western Blotting, also known as immunoblotting, a technique used to identify a specific protein in a sample containing multiple proteins. Discover how proteins are separated by gel electrophoresis and detected using specific antibodies to provide information on molecular weight and quantity.