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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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.