Bicinchoninic acid bca protein assay

Purified VLPs were quantified either by p24 ELISA (Innotest HIV antigen mAb, Fujirebio) following manufacturer’s instructions or by western blot. For western blot quantification, recombinant Gag protein [38 (link)] was used as standard. The standard curve started at 125 ng with 1:2 dilutions until 7.8 ng. Samples were treated as described above. Samples were denatured at 95 °C for 5 min, and proteins were separated by SDS-PAGE. After blocking, membranes were incubated with primary antibody anti-HIV-1 p24 antibody (Abcam, 1:2000) and secondary antibody Peroxidase AffiniPure Donkey anti-Mouse IgG (H+L, Jackson ImmunoResearch, 1:10,000).
The total protein content in the sample was assessed by Bicinchoninic Acid (BCA) Protein Assay (ThermoFisher Scientific).

Human granulocytes or erythrocytes were purified from healthy donor peripheral blood using a pan-granulocyte negative selection kit (Stemcell Technologies) or centrifugation on a Ficoll layer, respectively. Frozen human hepatocytes were purchased from XenoTech (Kansas City, KS). Lysates were prepared by microtip sonication followed with clarification by centrifugation. Plasma samples from renal cell carcinoma (RCC) patients were obtained by Ficoll centrifugation of whole blood purchased from Conversant Biologics (Huntsville, AL). Granulocyte lysate was assayed at 0.094 mg/mL, as determined by bicinchoninic acid/BCA protein assay (ThermoFisher), in reaction buffer. Erythrocyte or hepatocyte lysates were assayed at concentrations empirically determined to consume 10–15% of ¹³C(6)-L-arginine in 30 min at 37 °C. Arginase activity was determined in lysates and plasma by quantification of the generation of ¹³C(5)-L-ornithine from ¹³C(6)-L-arginine in the presence of a dose-titration of CB-1158.

Immunoblot analyses of cardiac tissue samples were carried out using a semi-quantitative western blotting analysis. The antibody used were: 1:1,000 rabbit anti-Ser^176/180-IKKα/β, 1:1,000 rabbit anti-total IKKα/β, 1:1,000 mouse anti-Ser^32/36-IκBα, 1:1,000 mouse anti-total IκBα, 1:1,000 rabbit anti-NF-κB, 1:1,000 rabbit anti-total BTK, 1:1,000 rabbit anti-Tyr¹²¹⁷ PLCγ, 1:1,000 rabbit anti-total PLCγ (from Cell Signaling), 1:1,000 rabbit anti-Tyr²²³-BTK, 1:5,000 rabbit anti NLRP3 inflammasome (from Abcam), 1:1,000 mouse anti-caspase 1 (p20) (from Adipogen). The apex of the heart was taken and homogenized in 1:10 of homogenization buffer at 4°C. Nuclear and cytosolic proteins were then extracted as previously described (21 (link)) and concentrations were quantified by bicinchoninic acid (BCA) protein assay (Thermo Fisher Scientific Rockford, IL). Proteins were separated by 8% sodium dodecyl sulfate (SDS)-PAGE and transferred to polyvinyldenedifluoride membranes. Membranes were blocked in 10% milk solution with TBS-Tween and then incubated with the primary antibody overnight at 4°C. The next day the secondary antibody was added for 30 min at room temperature and visualized using the ECL detection system. Tubulin and histone 3 were used as loading control. The immunoreactive bands were analyzed by the Bio-Rad Image Lab Software™ 6.0.1 and results were normalized to the sham bands.

Cecal tissue was homogenized by bead beating with buffer consisting of 1 M HEPES and Halt protease inhibitor cocktail (Thermo Fisher Scientific Inc., Rockford, IL) and then kept on ice for 30 min with buffer containing Triton X-100, HEPES, and Halt protease inhibitor cocktail. The homogenate was then spun at 10,000 × g for 10 min, and the supernatant was collected for cytokine protein measurement. Cytokines measured by ELISA (R&D Systems) included IL-4, IL-5, IL-9, IL-23, IL-17, IL-22, IL-25, and TNF-α. Cytokine concentrations were normalized to total protein concentration (bicinchoninic acid [BCA] protein assay; Thermo Fisher).

Blood samples were collected every 4 weeks from 7 weeks of age. Serum C3 was detected by ELISA (ICL lab, Portland, OR, USA), anti-double stranded DNA (dsDNA) antibody titers were quantified by radioimmunoassay and serum alanine transaminase (ALT), creatinine and blood cell counts were analyzed by using an autoanalyzer.
To determine kidney injury in MRL/lpr mice, urine samples were collected every 3 weeks from the 7 weeks of age by placing the mice individually in metabolic cages for 24 h. Urine protein concentrations were measured by bicinchoninic acid (BCA) protein assay (Thermo Fisher, Waltham, MA, USA).
For histological analyses, kidneys were fixed in paraformaldehyde, embedded in paraffin, stained with H&E and evaluated by a renal pathologist, who was blinded to the treatments of the mice. Glomerular injury was scored from 0 to 4 for each glomerulus and each sample involved evaluation of 30 glomeruli, within at least three separate parts of one section. The average of these 30 values was calculated as the injury score of an individual sample.

Bovine serum albumin (BSA) and lysozyme (LSZ) from chicken white egg were obtained from Sigma. Nanoparticles were dispersed in 0.05% BSA or LSZ water solution at three concentrations: 2 mg/mL, 4 mg/mL and 8 mg/mL. After 180 s of bath sonication, the samples were incubated for 4 h at 37 °C under constant agitation. Afterwards, samples were centrifuged (10,000× g, 20 °C, 15 min), and the remaining protein concentration was measured in the supernatant using bicinchoninic acid (BCA) protein assay according to the manufacturer’s instruction (ThermoScientific). An additional standard curve for lysozyme was prepared. The results were normalized to blank samples (0.05% BSA or LSZ). The experiment was performed in triplicates.

Either 1 × 10⁶ human bone marrow stromal cells (hBMSCs; n = 7 different donors) or whole hES-derived chondrocytes on membranes (n = 6–9 batches) were lysed with 500 μL of 2X Lysis buffer (Ray Biotech) supplemented with a phosphatase/protease inhibitor (Thermo-Fisher). Lysates were then centrifuged to remove cellular debris, and a Bicinchoninic acid (BCA) protein assay (Thermo-Fisher) was performed to quantify total lysed protein. ELISAs for FGF-2 (Ray Biotech), BMP-2 (R & D Systems), and TGF-β1 (R & D Systems) were performed according to the manufacturer’s protocols.