Understanding Western Blot Technique

Study Notes

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.

Description

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.