4 for 30 minutes at 20 u00b0 c the activation mixture was then loaded onto a 1 0 u00d7 10 cm monoq column pharmacia and eluted with a linear 20 column volume gradient to 1 0 m nacl in buffer b the activated p38 eluted after the adp and atp the activated p38 peak was pooled and dialyzed against buffer b 0 2 mm na 2 vo 4 to remove the nacl the dialyzed protein was adjusted to 1 1 m potassium phosphate by addition of a 4 0 m stock solution and loaded onto a 1 0 u00d7 10 cm hic rainin hydropore column previously equilibrated in buffer d 10 glycerol 20 mm u03b2 glycerophosphate 2 0 mm dtt 1 1 mk 2 hpo 4 the protein was eluted with a 20 column volume linear gradient to buffer d 50 mm k 2 hpo 4 the double phosphorylated p38 eluted as the main peak and was pooled for dialysis against buffer b 0 2 mm na 2 vo 4 the activated p38 was stored at u2212 70 u00b0 c this assay was carried out in the presence of 10 mm mgcl 2 25 mm u03b2 glycerophosphate 10 glycerol and 100 mm hepes buffer at ph 7 6 for a typical ic 50 determination a stock solution was prepared containing all of the above components and activated p38 5 nm the stock solution was aliquotted.