Epoxy activated sepharose 6b

ent-Sauchinone was conjugated with cyanogen bromide Epoxy-activated Sepharose 6B (Sigma, St Louis, Missouri, United States). Briefly, ent-Sauchinone (1 mg) was dissolved in 1 ml of coupling buffer (0.1 M NaHCO3 and 0.5 M NaCl, pH 6.0). The Epoxy-activated Sepharose 6B was swelled and washed in 1 mM HCl on a sintered glass filter, then washed with a coupling buffer. Epoxy-activated Sepharose 6B beads were added to the ent-Sauchinone containing coupling buffer and incubated at 4°C for 24 hours. The ent-Sauchinone-conjugated Sepharose 6B was washed with three cycles of alternating pH wash buffers (buffer 1: 0.1 M acetate and 0.5 M NaCl, pH 4.0; buffer 2: 0.1 M TriseHCl and 0.5 M NaCl, pH 8.0). ent-Sauchinone-conjugated beads were then equilibrated with a binding buffer (0.05 M TriseHCl and 0.15 M NaCl, pH 7.5). The control unconjugated Epoxy-activated Sepharose 6B beads were prepared as described above with the absence of ent-Sauchinone. The cell lysate or STAT3 recombinant protein (Abnova, Taipei, Taiwan) were mixed with ent-Sauchinone conjugated Sepharose 6B or Sepharose 6B at 4 C for 24 hours. The beads were then washed three times with TBST. The bound proteins were eluted with SDS loading buffer. The proteins were then resolved by SDS-PAGE followed by immunoblotting with antibodies against STAT3 (1:1000 dilution, Santa Cruz Biotechnology Inc.).

BV2 cell lysate was conjugated with epoxy-activated sepharose 6B (Sigma-Aldrich, St. Louis, MO). iNOS (1 mg) was dissolved in 1 mL of coupling buffer (35% DMSO and 0.5 M NaCl, pH 8.3). The epoxy-activated sepharose 6B was swelled and washed in 1 mM HCl through a sintered glass filter, then washed with a coupling buffer. epoxy-activated sepharose 6B beads were added to the MCZ-containing coupling buffer and incubated at 4 °C for 24 h. The iNOS-conjugated sepharose 6B was washed with three cycles of alternating pH wash buffers (buffer 1, 0.1 M acetate and 0.5 M NaCl, pH 4.0; buffer 2, 0.1 M Tris-HCl and 0.5 M NaCl, pH 8.0). iNOS-conjugated beads were then equilibrated with a binding buffer (0.05 M Tris-HCl and 0.15 M NaCl, pH 7.5). The control unconjugated epoxy-activated sepharose 6B beads were prepared as described above in the absence of iNOS. The cell lysate was mixed with iNOS-conjugated sepharose 6B or sepharose 6B at 4 °C for 24 h. The beads were then washed three times with Tris-buffered saline and Tween 20 (TBST). The bound proteins were eluted with SDS loading buffer. The proteins were then resolved by SDS-PAGE followed by immunoblotting with antibodies against iNOS (1:1000, Novus Biologicals, Inc., Littleton).

Half gram Epoxy-activated sepharose-6B (GE Healthcare Life Sciences) was swollen in 50 mL distilled water for 1 hour, and 50 ml coupling buffer (0.1 M Na₂CO₃, pH 13.0 or pH 9.5) was applied for washing beads through a sintered glass filter. Lactic acid (Sigma-Aldrich) (0.5 g in 15 ml coupling buffer) was mixed with Epoxy-activated sepharose-6B, the pH adjusted to either 9.5 or 13.0, and the coupling allowed for 16 h at 37 °C with gentle shaking. The beads were washed to get rid of free ligand with coupling buffer, and remaining active chemical groups blocked by 1 M ethanolamine at pH 8.0. The amounts of cross-linked ligand were much higher in the 6B-LAC-2 (pH 13.0) than the 6B-LAC-1 (pH 9.5) resins (text). The beads were preserved in a solution containing 20% ethanol until use. Before use, ethanol was washed off, and the beads were incubated with cell lysates for 4 h, washed 4 times with HEPES-lysis buffer (50 mM HEPES-KOH, pH7.4, 180 mM NaCl, 1.5 mM MgCl₂, 5 mM EDTA, 10% glycerol, 1% NP-40, 100X NaVO₃), and re-suspended with HEPES-lysis buffer for Western-Blot and mass spectrometry analyses.