Nampt (Visfatin/PBEF) (human) (rec.) (His)

Figure 2: Dimer formation of recombinant Nampt (Visfatin, PBEF) (AG-40A-0018). Purified Nampt (Visfatin, PBEF) was separated by SDS-PAGE and Western blot analysis was performed using rabbit anti-Nampt polyclonal antibody (AG-25A-0025). In the absence of DTT, Nampt (visfatin, PBEF) formed a homodimer, a homomultimer.

Figure 3: Measurement of NAMPT enzymatic activity was performed as described previously [G.C. Elliott, et al.; Anal Biochem 107, 199 (1980)]. The recombinant Nampt was diluted in assay buffer and 10μl per 50μl reaction mix were applied in the reaction mix (20 mmol/l TrisHCl pH 7.4; 2,5 mmol/l ATP; 50 mmol/l NaCl; 12,5 mmol/l MgCl2; 2 mmol/l DTT; 0,5 mmol/l PRPP; 5 μmol/l 14C-nicotinamide) and incubated at 37°C for 1h. The 50μl reaction mix was transferred into tubes containing 2ml of acetone and afterwards pipetted onto acetone-presoaked glass microfibre filters (GF/A Ø 24 mm). After rinsing with 2 x 1ml acetone, filters were dried, transferred into vials with 6ml scintillation cocktail and radioactivity of 14C-NMN was quantified in a liquid scintillation counter. After subtraction of buffer values as background, cpm were normalized to 10⁶ cells and the volume of enzyme preparation (10μl). Mouse liver lysate at a concentration of 34.5μg/ml was used as positive control in each assay. The positive control is mouse liver lysate at a concentration of 34,5μg/ml – normally, which brings the most counts per minute (cpm) (contributed by Antje Garten & Dr. Kiess, University of Leipzig, Germany).