Western Blots

A Western blot is a technique used to detect and analyze specific proteins in a sample. It combines gel electrophoresis with antibody-based detection.

How it works:

Proteins are first separated by size using SDS-PAGE, a type of gel electrophoresis that uses a detergent to denature proteins and give them a uniform negative charge. The proteins are then transferred from the gel to a membrane, typically made of nitrocellulose or PVDF. The membrane is incubated with a primary antibody that specifically binds to the target protein. After washing away unbound antibodies, a secondary antibody is added. This secondary antibody binds to the primary antibody and is linked to an enzyme. The membrane is then exposed to a substrate that reacts with the enzyme to produce a visible signal—often a color change or light emission—resulting in a band that marks the presence of the protein.

Applications:

• Detecting specific proteins in complex samples

• Measuring protein expression levels

• Confirming the presence of proteins after RNAi or CRISPR editing

• Studying post-translational modifications

How to interpret data:

Western blot results are visualized as bands. The presence, intensity, and size of the bands indicate whether the target protein is present, how abundant it is, and whether it is the expected size. Comparisons can be made between samples to assess differences in expression.