Western blot protocol pdf

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In a dot blot the biomolecules to be detected are not first separated by electrophoresis. Instead, a mixture containing the molecule to be detected is applied directly on a membrane as a dot, and then is spotted through circular templates directly onto the membrane or paper substrate. This differs from the western blot because protein samples are not separated electrophoretically.

The technique offers significant savings in time, as chromatography or gel electrophoresis, and the complex blotting procedures for the gel are not required. However, it offers no information on the size of the target biomolecule. Furthermore, if two molecules of different sizes are detected, they will still appear as a single dot. DNA probes or the antibody.

Because a dot blot does not require any specific instruments, lots of dot blot assays are developed using antibodies with high specificity to detect different protein targets. Dot blot is also used to evaluate or screen the effectiveness of the antibodies. The following is a general outline of an antigen-antibody-antibody dot blot assay protocol.

Specific concentrations need to be determined for each assay. Other types of dot blot assays, such as antibody-antigen-antibody can be performed in a similar fashion.

The dot blot was originally performed on a piece of nitrocellulose membrane or PVDF membrane. After the protein samples are spotted onto the membrane, the membrane is placed in a plastic container and sequentially incubated in blocking buffer, antibody solution, or rinsing buffer on a shaker. Finally, for chemiluminescence imaging, the piece of membrane is wrapped in a transparent plastic film filled with enzyme substrate.

For chemiluminescence signal detection, the apparatus is dissembled and the membrane is taken out and wrapped in a transparent plastic film filled with enzyme substrate. Vitrozm’s 96 well Zoom Blot method uses an absorption plug to wick away the solution from the membranes, which are individually assembled in each well. The Zoom Plate is a disposable self-standing device, without any external vacuum or set up apparatus, which allows dot blots to be performed much easier and faster.

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