Coomassie plus protein assay

Concentrations of lactate dehydrogenase (LDH) and γ-glutamyl transferase (GGT) were determined using commercially available kits (Thermo Scientific, Middletown, VA). Albumin and total protein concentrations were measured by the SPQ test system (DiaSorin, Stillwater, MN) and the Coomassie plus protein assay (Pierce Chemical, Rockford, IL) with a standard curve prepared with bovine serum albumin (Sigma), respectively. Activity of N-acetyl-β-D-glucoaminidase (NAG) was determined using a NAG assay kit (Roche Applied Science, Indianapolis, IN). All biochemical assays were modified for use on the KONELAB 30 clinical chemistry spectrophotometer analyzer (Thermo Clinical Lab Systems, Espoo, Finland) as described previously [46 (link)]. Concentrations of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and macrophage inhibitory protein-2 (MIP-2) in BALF were determined using commercial multiplexed fluorescent bead-based immunoassays (Milliplex Map Kit, Millpore Co., Billerica, MA) measured by a Luminex 100 (Luminex Co., Austin, TX) following the manufacturer’s protocol. The limits of detection (LOD) of each cytokine were 6.27, 3.28 and 29.14 pg/mL for TNF-α, IL-6 and MIP-2, respectively, and all values below these lowest values were replaced with a fixed value of one-half of the LOD value.

Concentrations of lactate dehydrogenase (LDH) and γ-glutamyl transferase (GGT) were determined in BALF using commercially available kits (Thermo Scientific, Middletown, VA). Albumin and total protein concentrations were measured by the SPQ test system (DiaSorin, Stillwater, MN) and the Coomassie plus protein assay (Pierce Chemical, Rockford, IL) with a standard curve prepared with bovine serum albumin (Sigma), respectively. Activity of N-acetyl-β-D-glucoaminidase (NAG) was determined using a NAG assay kit (Roche Applied Science, Indianapolis, IN). All assays were modified for use on the KONELAB 30 clinical chemistry spectrophotometer analyzer (Thermo Clinical Lab Systems, Espoo, Finland) as described previously [31 (link)].

Most of the plasmid constructs and protein expression procedures have been described^{7 (link)}. Briefly, CyPet-SUMO1 and YPet-Ubc9 were cloned into the NheI/NotI sites of pET28(b) vector (Novagen). BL21(DE3) Escherichia coli cells were transformed with pET28 vectors encoding CyPet-SUMO1 or YPet-Ubc9. The expression of Poly-his tagged recombinant proteins was induced with 0.1 mM IPTG at 25 °C overnight. The recombinant proteins were then purified by Ni²⁺-NTA agarose beads (QIAGEN) and eluted by buffer containing 20 mM Tris-HCl, pH 7.5, 200 mM NaCl, and 150 mM imidazole. After the proteins were dialyzed in buffer containing 20 mM Tris-HCl, pH 7.5, 50 mM NaCl, and 1 mM DTT, they were concentrated and purified by gel filtration HPLC with Superdex75 10/300 GL column with a HPLC purification system (ÄKTA^TM purifier. GE Healthcare). Purity of proteins was confirmed by SDS-PAGE and Coomassie blue staining, and concentrations were determined by Coomassie Plus Protein Assay (Thermo-Fisher).