Penta·His Antibody, BSA-free (100 ug)

Protocol tips

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	Use 0.1 μg/ml as working concentrattion

Protocol tips
Use 0.1 μg/ml as working concentrattion

Publication protocol

Protein analysis: Conditioned medium from Epo-expressing cell lines and fractions from all column elution steps were characterized by western blotting, using anti-human Epo monoclonal antibody AE7A5 [20]. Thirty to fifty μl of sample per lane were subjected to denaturing gel electrophoresis, followed by electrophoretic transfer [22] to PVDF membrane (Millipore). Highly purified recombinant human Epo (non His-tagged Epowt, rhEpo; gift of Elanex Pharmaceuticals, Bothell, WA) was used as a positive control. Blots were incubated in the presence of 0.25 μg AE7A5/ml, and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology) was employed as second antibody. Chemiluminescent detection was performed using Super Signal (Pierce).
The presence of His6-tagged Epo in COS-7 CM, in CHO CM, and in samples at each step of the purification process was monitored using an anti-His tag antibody. Both standard SDS-PAGE/western blotting (to detect denatured protein) and dot blotting were used. For dot blot analysis, samples were applied directly to nitrocellulose membrane, dried, and fixed by baking at 80ºC. Murine dihydrofolate reductase (DHFR) with a C-terminal His6-tag (vector pQE-16, Qiagene) and human selenium binding protein (hSP56) with an N-terminal His6-tag prepared in our laboratory, both purified on Ni2+-NTA resin, were used as positive controls. Following appropriate blocking, blots were incubated in the presence of one of the following anti-His monoclonal antibodies: anti-polyHistidine (Sigma), penta-His antibody, tetra-His antibody or RSG-His antibody (all from Qiagen), using the antibody concentrations recommended by the manufacturers (Table 1). HRP-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology) was employed as second antibody, and chemiluminescent detection was performed using Super Signal (Pierce).

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Manufacturer protocol

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