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PhosphoProtein Purification Kit

For purification of phosphorylated proteins by affinity chromatography
  • Complete separation of phosphorylated and unphosphorylated proteins
  • One kit contains columns, buffers, reagents, and ultrafiltration columns
  • Cell-signaling studies without the need for radioactivity

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.

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Cat No./ID: 37101
PhosphoProtein Purification Kit (6)
6 PhosphoProtein Purification Columns, 6 Nanosep Ultrafiltration Columns, Reagents, Buffers
The PhosphoProtein Purification Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

Product Details

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.

Yields of phosphorylated proteins obtained using the PhosphoProtein Purification Kit.
Cell type Number
of cells
processed
Total protein in cell lysate (µg) Protein loaded
onto column (µg)
Protein
in eluate (µg)
Percentage
phosphorylated proteins
CHO 1.5 x 107 3400 2500 300 12%
NIH 3T3 n.d. 2750 2500 165 7%
293 1.5 x 107 3650 2500 200 8%
Cos-7 4.5 x 106 1700 1700 120 7%
Huh-7 8.5 x 106 2650 2500 235 9%
HT 29 n.d. n.d. 2500 200 8%
LT 23 n.d. n.d. 2500 275 11%
HeLa S3 1.8 x 107 5950 2500 280 11%
HeLa Acc57 6.6 x 106 2500 2500 235 9%

Applications

The PhosphoProtein Purification Kit provides complete separation of phosphorylated and unphosphorylated protein fractions for research in:

  • Proteomics 
  • Cell signaling and apoptosis
  • Protein kinases and oncology
  • Immune disorders

Specifications

Features
Specifications
Applications SDS-PAGE, mass spectrometry
Binding capacity/yield 500 µg
Fractions isolated Two fractions
Protein with post-translational modification Phosphorylation
Sample size ~1 x 10e7 cells
Species Eukaryotes
Start material Cells

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Kit Handbooks (2)

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For purification and detection of phosphorylated
proteins from eukaryotic cells and tissues
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