Protein quantification assay kit

Total protein was extracted from the cell pellets using the NucleoSpin RNA/Protein Kit (Macherey‐Nagel, Dueren, Germany), and concentration determined using the Protein Quantification Assay Kit (Macherey‐Nagel) and a microplate photometer (Multiskan EX, Thermo Scientific, Waltham, MA, USA). Equal amounts of protein (10 μg) were resolved by SDS‐PAGE. Band intensity, mirroring protein levels were visualized using chemiluminescence detection systems Supersignal West Pico (Thermo Scientific) or Substrat HRP Immobilon Western (Merck Millipore) following the manufacturers’ instructions.

Proteomic analyses were performed at the Proteomics Unit of the University of Valencia (SCSIE), Valencia, Spain (PRB2-ISCIII ProteoRed Proteomics Platform). Proteins from spermatozoa were extracted using Ringer’s lysis solution (22 g/L urea and 9 g/L NaCl). After mixing with lysis buffer, samples were sonicated for 5 min and centrifuged for 5 min at 15,870× g at 4 °C and diluted 1 to 10 with 50 mM ABC. The protein concentration from every sample was determined by a Protein Quantification Assay Kit (Macherey-Nagel, Düren, Germany).

E. coli BL21(DE3)(pET-AgaB-4) cells were cultured in 1 L of LB broth containing kanamycin (30 μg/mL), with shaking at 37 °C. Cells were cultured to an OD₆₀₀ of 0.4–0.6. Subsequently, IPTG (0.1 mM) was added to induce rAgaB-4 expression at 20 °C for 24 h. Cells were harvested by centrifugation at 8000×g for 30 min and were then resuspended in lysis buffer. The cell suspension was disrupted using Constant Cell Disruption Systems (Constant Systems Ltd, Warwick, UK). The cell lysate was centrifuged at 8000×g for 15 min at 4 °C, and the resulting supernatant was filtered through a 0.22-μm membrane and applied to a 5-mL HiTrap™ excel affinity chromatography column (GE Healthcare, Uppsala, Sweden) according to the manufacture’s instruction. The purity of the eluted fusion protein was analyzed through 12.5% SDS-PAGE, and the protein concentration was determined using a Protein Quantification Assay Kit (MACHEREY–NAGEL, Düren, Germany).

The construction of EGF-TiO₂ PEG NPs is shown in Figure 1. Recombinant human epidermal growth factor at 0.2 mg/mL (EGF, Thermo Fisher Scientific, Waltham, MA, USA) was mixed with 0.5% (w/v) TiO₂ PEG NPs in a 20 mM HEPES buffer solution (pH 7.4) at 4 °C overnight. The mixture was centrifuged at 14,000× g for 30 min and reconstituted with endotoxin-free sterilized water. This substitution process was repeated three times in order to remove the non-absorbed EGF, then the EGF-conjugated TiO₂ PEG NPs were collected and sonicated for 15 min. The EGF amount of the supernatants of the EGF-TiO₂ PEG NPs after centrifugation was measured to determine the conjugation efficiency by using a protein quantification assay kit (Macherey-Nagel GmbH & Co. KG, Düren, Germany). The conjugation efficacy was more than 90% of the initial amount of EGF. The evaluation of average hydrodynamic particle size in water was carried out by dynamic light scattering. EGF would be attached on the TiO₂ PEG NPs surface by physical adsorption.

Proteins and RNA were extracted from the same biological samples using the Nucleospin RNA/protein kit (Macherey Nagel, France). Proteins were quantified using the Protein Quantification Assay kit (Macherey Nagel, France), according to the manufacturer’s instructions. Western blot analyses were performed as described previously [29 (link)] except that blots were blocked for 1h at RT in PBS-0.1%Tween-20-5% dry milk.

Whole cell proteins from three colon cancer cell lines (SW-480, SW-48 and HCT116) and one non-cancer colon epithelial cell line (FHC) were used in this study. All the cell lines purchased from ATCC (American type culture collection). SW-480 and SW-48 were maintained in RPMI (Roswell Park Memorial Institute) 1640 medium (Life Technologies Australia Pty Ltd, Melbourne, VIC, Australia) containing 10% fetal bovine serum (FBS) (Life Technologies Australia Pty Ltd) and 1% penicillin/streptomycin (Life Technologies Australia Pty Ltd) at 37 °C in a CO₂ incubator. HCT116 cells were cultured and maintained in DMEM (Dulbecco’s Modified Eagle’s Medium) medium (Life Technologies Australia Pty Ltd) modified with 10% FBS and 1% penicillin/streptomycin at 37 °C in a CO₂ incubator. FHC cells were cultured in DMEM: F-12 medium (1:1) (Life Technologies Australia Pty Ltd) supplemented with 10 ng mL⁻¹ cholera toxin (Sigma-Aldrich), 0.005 mg mL⁻¹ insulin (Life Technologies Australia Pty Ltd), 100 ng mL⁻¹ hydrocortisone (Sigma-Aldrich), 0.005 mg mL⁻¹ transferrin (Life Technologies Australia Pty Ltd) and 10% FBS 37 °C in a CO₂ incubator. Total proteins were extracted from all cultured cells with lysis buffer (Bio-Rad, Gladesville, NSW, Australia) and subsequently were quantitated by absorbance spectrometry using protein quantification assay kit (Macherey-Nagel, Bethlehem, PA, USA).