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.
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.
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.
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.)