Western Blot - 2. Types of Blot Formats

Western blot experiments can be performed in several formats, most requiring a conjugated secondary antibody to act as the reporter molecule. Reporter molecules include enzymes(peroxidase, alkaline phosphatase) and fluorochromes.

Enzymes require substrate that allows for color or light to be generated. The data can be detecting on a scanner, using film, or a commercial imaging system.

Fluorochrome reporter molecules do not require substrate, but do require specialized equipment with light filters for data collection. Western blot experiments immobilize protein analyte onto a membrane. The choice of membrane depends on the type of experiment to be performed.  Most commonly used are nitrocellulose or polyvinyldifluoride (commonly PVDF). Nitrocellulose is easy to use and provides suitable data for most common enzymatic reporter experiments. Low fluorescent PVDF membranes are recommended for fluorescent western blot applications. A schematic illustration of 2 common western blot formats is shown in the figure to the left.

Western Blot Protocol

1. Background
2. Types of Western Blot Formats
3. Primary Antibody Reagents
4. Secondary Reporter Antibody Reagents
5. Substrates
6. Buffers and Blocking Reagents
7. Western Blot Preassembled Kits