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What is western blot
There are many Blottechniques in molecular biology such as southern blot,
northern blot, eastern blot and western blot. The principle and process of these blot
techniques are similar. Southern and northern blot are nucleic acid blotting
techniques, of which southern blot is for detecting a specific DNA sequence in DNA
samples and northern blot is to study gene expression by detection of RNA (or
isolated mRNA) in a sample. Eastern blot is used to analyze protein post translational
modifications (PTM) such as lipids, phosphomoieties and glycoconjugates.
Different from those blot technique described above, western blot, also called
protein immunoblot, is an analytical technique used to detect specific proteins in a
given sample such as cell lysates, tissue homogenate or extract. It is widely used in
molecular biology.
The bases of western blot identification are two distinguishing properties: molecular
weight and antibody binding specificity. It uses gel electrophoresis (usually SD S-PAGE)
to separate native proteins by 3-D structure or denatured proteins by the length of
the polypeptide. And then transferred the protein sample to a membrane (typically
nitrocellulose or PVDF), where they are probed with labeled-antibodies and stained
with dyes for visualization and directly identified by N-terminal sequencing, mass
spectrometry or immunodetection.
Immunodetection involves the identification of a protein through its reaction with a
specific antibody. Through spatial resolution, this method provides molecular weight
information on individual proteins and distinguishes isoforms, alternate processing
products, and other post-translationally modified forms.