Mouse anti his tag

Human epithelial kidney (HEK) cells grown to confluence in 10 cm plates were transfected with 5 μg of mammalian expression plasmids (pcDNA3.1(+), Thermo Fisher, cat #V790-20, Waltham, MA) that express either wild-type EPO or EPO-R76E with carboxyl-terminal His-tags. A 19-amino acid linker containing a Tobacco Etch Virus (TEV) protease recognition site was inserted after the carboxyl-terminus of EPO-R76E to increase the accessibility of the His-tag to the metal ion matrix used for purification. The next day, plates were washed and replaced with serum-free DMEM. After an additional three days, the media was collected and centrifuged to pellet cells. Supernatant was applied to a TALON cobalt resin column (Takara BIO, Mountain View, CA) and washed with 20 bed volumes of phosphate buffered saline (PBS). Protein was eluted from the matrix with 50 mM potassium acetate, pH 5.0 buffer containing 100 mM NaCl. The pH of the solution was neutralized with a 1:40 volume (2.5 μ100 μl eluate) of 0.8 M Tris, pH 9.3. Protein was judged to be at least 90% pure based on gels stained with SimplyBlue SafeStain (Invitrogen, Carlsbad, CA). His-tagged EPO or EPO-R76E was detected on blots with mouse anti-His tag (Cell Signaling, Danvers MA; #2366 diluted 1:1000) or rabbit anti-hEPO (R&D Systems Inc., Minneapolis MN; #AB286 at 1 μg/ml).