The Phosphoprotein Enrichment Kit provides an effective affinity-based procedure for isolating phosphorylated proteins from mammalian cells and tissues. Each kit includes a complete set of buffers along with six high-capacity columns for enrichment of both cytosolic and membrane-bound phosphoproteins, regardless of the amino acid modified—including serine, tyrosine, or threonine.

Our enrichment procedure offers a number of advantages:

• The procedure is fast; the average cell-to-sample purification time is less than 2 hours.
• It is also straightforward, consisting of four main steps: adding Extraction/Loading Buffer to the cell or tissue pellet to extract total cellular protein, loading the extract on an affinity column, washing, and finally eluting the bound phosphoprotein with a detergent-free Elution Buffer.
• A single buffer—Extraction/Loading Buffer—is used for both the protein-extraction and affinity-column steps, making buffer exchange unnecessary. This saves time and prevents sample loss.
• The procedure is nondenaturing, so phosphoproteins remain folded throughout the process, even during the extraction and elution steps.

Each column has a maximum binding capacity of ~4 mg of phosphorylated protein.

Highly selective enrichment of phosphoproteins

The Phosphoprotein Enrichment Kit may be used with any mammalian cell type. Cell lines tested include NIH 3T3, HEK 293, HeLa, Cos-7, and Jurkat. The enrichment procedure is highly efficient, as demonstrated by Western blotting analyses. Using a colorimetric phosphate detection method, we found the majority of the phosphoprotein in the eluate; negligible traces were detected in the wash fraction.

Phosphoprotein Affinity Columns yield a concentrated solution of phosphoprotein that can be analyzed by several different methods, including mass spectrometry and two-dimensional polyacrylamide gel electrophoresis (2D-PAGE).