Deae sephadex

Chemicals, biochemicals, buffers, and solvents were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO), Fisher Scientific Inc. (Pittsburgh, PA), Fluka Chemical Corp. (Milwaukee, WI), or EMD Millipore, Inc. (Billerica, MA). 2-Hydroxymuconate (2-HM)¹¹ and 3-bromopropiolate (3-BP)¹² were synthesized by the indicated references. Phenylenolpyruvate (PP) was purchased from Fluka Chemical Corp. (Milwaukee, WI) as the crystalline free acid, which exists exclusively in the enol form. Propiolic acid (98%) was purchased from Sigma-Aldrich, and purified further prior to use by distillation. N2 was purified by a literature procedure.^{13 (link)} N2 is an enzyme from Gammaproteobacteria bacterium SG8_31 (UniProt accession A0A0S8FF56) and is in the 4-oxalocrotonate tautomerase (4-OT) subgroup, as described elsewhere.^{13 (link)} The DEAE Sephadex, Phenyl-Sepharose 6 Fast Flow, and Sephadex G-75 resins were obtained from GE Healthcare Bio-sciences (Pittsburgh, PA). The Econo-Column^® chromatography columns were obtained from BioRad (Hercules, CA). The Amicon stirred cell concentrators and the ultrafiltration membranes (3,000 or 5,000 Da, MW cutoff) were purchased from EMD Millipore Inc.

Frozen recovered human plasma was obtained from volunteer donors from the San Diego Blood Bank. DEAE-Sephadex and Sephacryl S300 were from GE Healthcare (Little Chalfont, UK). 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes), Polyethylene Glycol 4000 (PEG), (N-morpholino)ethane sulfonic acid (Mes), (N-morpholino)propanesulfonic acid (Mops), Horseradish Peroxidase (HRP), 3,3′,5,5′-tetramethylbenzidine (TMB) and 3-amino-9-ethylcarbazole were from Sigma-Aldrich Chemical Corp. (St. Louis, MO). Luminata forte chemoluminescent HRP detection reagent for ELISA was from EMD/Millipore (Billerica, MA). A murine mAb (clone B-5) directed against the amino terminal modules of human Fibulin-1 was purchased from Santa Cruz Biotechnology (Dallas, TX). Goat anti-rabbit IgG coupled to HRP was purchased from Life Technologies (Carlsbad, CA). Factor H was purified from human plasma similarly to a published method using the following steps: 4–12% PEG precipitation of barium citrate treated plasma, DEAE-Sephadex column chromatography, gel filtration through Sephacryl S300, and column chromatography on dextran sulfate Sepharose [29 (link)]. Factor H-Sepharose was prepared by conjugating Factor H (10 mg/ml) in 0.15 M NaHCO₃ pH 8.2 to Sepharose 6B using the cyanogen bromide reaction [30 ].