Phosphoprotein Enrichment Kit

The Phosphoprotein Enrichment Kit provides an effective affinity-based procedure for isolating phosphorylated proteins from mammalian cells and tissues (Figure 1). 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 (Figure 2): 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. Each column has a maximum binding capacity of ~4 mg of phosphorylated protein, and the procedure is nondenaturing, so phosphoproteins remain folded throughout the process, even during the extraction and elution steps.

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 (Figure 1). 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).