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Protein Phosphorylation Identification Methods

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#1 larry



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Posted 22 September 2018 - 11:25 PM

Western Blotting
Western Blotting is an immunobiochemical analysis method based on gel electrophoresis and solid phase immunoassay technology to detect a certain protein in a complex sample according to specific antigen-antibody specific reaction.
Technical Advantages
• High detection specificity
• Ability to specifically identify individual phospho site
•Phosphorylation is limited by phosphorylated antibodies, therefore highly specific and specific phosphorylated antibodies are difficult to prepare. While, newly discovered phosphorylation sites often lack effective phosphorylation detection antibodies.
Isotope-labeled Western Blotting
The principle of this method is to identify the gelshift caused by the incorporation of radioactive 32P and the change of molecular mass when the protein is phosphorylated, and then detect the isotope label by Western Blotting to achieve the purpose of identifying the phosphorylated protein.
Technical Advantages
• Detection technology is sensitive and intuitive
• Commonly used for in vitro phosphorylation reaction detection
• Phosphorylation is not restricted by antibodies
• Simultaneous analysis of multiple protein phosphorylation
Mn2+-Phos-tag SDS-PAGE
SDS-PAGE allows the retention of phosphorylated proteins by the affinity of the dinuclear metal (such as Mn2+) complex attached to the acrylamide molecule to the phosphate group, and then transfers the electrophoresis results to the PVDF membrane. Protein recognition by the corresponding protein was used to detect protein phosphorylation based on migration retention.


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