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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.

Cat No./ID: 37101
PhosphoProtein Purification Kit (6)
$882.00
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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.
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Highly specific separation of phosphorylated proteins.
Non-stimulated Jurkat cells were radioactively labeled in vivo using 32P. The cell lysate was processed using the PhosphoProtein Purification Kit and the radioactivity in each fraction measured. (Data kindly provided by Gudrun Rehg and Sascha Dammeier, Byk Gulden, Konstanz, Germany.)

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Phosphoprotein purification procedure.
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Complete separation of unphosphorylated and phosphorylated proteins.
Complete Separation of Unphosphorylated and Phosphorylated Proteins. Protein-specific immunodetection of  [A]  unphosphorylated HSP-60 protein, and  [B]  phosphorylated p44 and p42 mitogen-activated protein kinase (MAPK) proteins. F: flow-through; E: eluate fractions. The antibody used to detect MAPK recognizes an epitope containing phosphorylated residues at Thr202 and Tyr204 in the p44 (upper band) and p42 (lower band) MAPK amino acid sequences. The absence of unphosphorylated HSP-60 in the eluate fraction and the absence of phosphorylated MAPK in the flow-through fraction demonstrate the complete separation of phosphorylated proteins using the PhosphoProtein Purification Kit.
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Efficient enrichment for 2D-PAGE phosphoproteome analysis

HeLa cells were treated with the phosphatase inhibitors  Calyculin (100 nM) and orthovanadate (1 mM) for 20 min at 37°C.    [A] After harvest and cell lysis, an aliquot of the lysate was separated by 2D-PAGE and proteins visualized using a non-specific total protein stain (blue) and a phosphoprotein-specific stain (orange, images overlaid).    [B] A second aliquot was processed using the PhosphoProtein Purification Kit and the eluate (containing phosphoproteins) treated as before. Overlapping protein spots are colored black. Image analysis indicated that 87% of eluted proteins are phosphorylated.

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SDS-PAGE analysis of flow-through and eluate fractions.

Flow-through fractions (left-hand lanes) and eluates (right-hand lanes) from cell lysates processed using the PhosphoProtein Purification Kit. Both fractions were concentrated by a factor of 10 before loading onto the gel. Proteins were visualized by Coomassie staining.

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
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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