PhosphoProtein Purification Kit
For purification of phosphorylated proteins by affinity chromatography
The PhosphoProtein Purification Kit, which is based on affinity chromatography, delivers a complete separation of phosphorylated and unphosphorylated proteins from a cell lysate, and therefore facilitates investigation of the phosphorylation status of both entire cells and specific proteins. In addition, the ratio of phosphorylated to unphosphorylated forms of proteins can easily be determined. The drastic reduction of the complexity of each fraction is especially useful when studying proteins of low abundance. The PhosphoProtein Purification Kit includes columns, reagents, buffers, and Nanosep Ultrafiltration Columns for efficient concentration and desalting of protein fractions.
The PhosphoProtein Purification Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
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Performance
The PhosphoProtein Purification Kit allows for highly specific separation of phosphorylated proteins and complete separation of unphosphorylated and phosphorylated proteins (see figures Highly specific separation of phosphorylated proteins and SDS-PAGE analysis of flow-through and eluate fractions). The PhosphoProtein Purification Kit is also applicable in proteome analysis (see figure Efficient enrichment for 2D-PAGE phosphoproteome analysis).
Principle
The PhosphoProtein Purification Kit is based on an affinity chromatography process and provides complete separation of phosphorylated and unphosphorylated proteins from a cell lysate, and therefore facilitates investigation of the phosphorylation status of both entire cells and specific proteins. Phosphorylated proteins can be identified after blotting using highly specific mouse monoclonal PhosphoThreonine and PhosphoSerine Antibodies, which recognize an epitope consisting of either a phosphothreonine or phosphoserine residue, irrespective of the surrounding amino acids. See figure Complete separation of unphosphorylated and phosphorylated proteins.
Procedure
Each PhosphoProtein Purification Column can be used to purify phosphorylated proteins from 107 eukaryotic cells (equivalent to approximately 2.5 mg total protein). Cells are lysed in the supplied lysis buffer, which contains a detergent and nucleases to resolve protein complexes, and protease inhibitors to prevent protein degradation. Cleared lysates are loaded onto the PhosphoProtein Purification Column, where phosphorylated proteins in the lysate bind to the column, while unphosphorylated proteins are found in the flow-through fraction. After a wash step, phosphorylated proteins are eluted from the column (see flowchart Phosphoprotein purification procedure ). Typically approximately 10% of the total protein loaded is in the phosphorylated fraction (depending on cell type and status). Both fractions retain biological activity and can be further purified if desired. Nanosep Ultrafiltration Columns are supplied with the PhosphoProtein Purification Kit to enable efficient concentration and desalting of protein fractions. The table shows typical protein yields in each fraction after separation using the PhosphoProtein Purification Kit.
Applications
The PhosphoProtein Purification Kit provides complete separation of phosphorylated and unphosphorylated protein fractions for research in:
Le site web n’est pas disponible en français dans son intégralité. Son contenu est international et peut aller à l’encontre des règlementations locales.
Le site web n’est pas disponible en français dans son intégralité. Son contenu est international et peut aller à l’encontre des règlementations locales.
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