PhosphoSolutions’ Protocols

Western Blotting | Lysate Preparation | Protein Dephosphorylation Methods

Western Blotting

Western blotting (WB) is the most widely performed immunoassay and is the best initial validation technique used to identify proteins of interest within a tissue homogenate or cell extract. In WB, proteins are first separated based on size (molecular weight) via gel electrophoresis. The resulting gel is then placed next to a membrane, made of nitrocellulose or polyvinylidene fluoride (PVDF), and the application of an electrical current induces the proteins to transfer from the gel to the membrane. The membrane can then be probed with antibodies that are specific for a target protein of interest. Lastly, the membrane is exposed to secondary antibodies and detection reagents enabling visualization.
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electrophoresis boxes

Lysate Preparation

How one prepares a lysate is crucial, especially when studying phosphorylated proteins.  Proteins rapidly dephosphorylate upon harvesting, so timing is critical when preserving phosphorylated proteins.  Along with timing, the detergents used in preserving phosphorylation are a key component.  When lysing is done properly, a large pool of lysed cells or organs can be prepared and stored in working volumes for long term studies.  Preparing pools for studies is an important component to consistent and reproducible results.
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Protein Dephosphorylation Methods

Phosphospecific antibodies are designed to differentiate between the phosphorylated and the non-phosphorylated states of a protein. The method to determine if or how well a phosphospecific antibody can distinguish between these different protein forms is evolving with new techniques using phosphatase enzymes. The protocols listed in Protein Dephosphorylation Methods show how one can dephosphorylate proteins when fixed to a membrane, while harvesting tissue culture cells, or after collecting animal organs. Additionally, protein dephosphorylation can be performed on frozen cells and organs if correctly preserved. Choosing the right phosphatase enzyme is crucial; knowing the specific activity and which amino acids the enzyme dephosphorylates are key components.
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