PhosphoProtein Purification Kit
- 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) $410.00 6 PhosphoProtein Purification Columns, 6 Nanosep Ultrafiltration Columns, Reagents, Buffers |
Product Details
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.
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.
| 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% |
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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