Proteojet membrane protein extraction kit

After the required treatments, the cells were scraped in phosphate-buffered saline and collected in a microfuge tube followed by centrifugation at 3000 rpm for 5 min at 4 °C. Cells were lysed in lysis buffer (150 mM NaCl, 20 mM Tris-HCl pH 8.0, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 mM NaF, 1 mM Na₃VO₄, 0.5% NP40, and protease/phosphatase inhibitors). Membrane and cytoplasmic proteins were obtained using the ProteoJET™ Membrane Protein Extraction Kit (Fermentas, Waltham, MA, USA) according to the manufacturer’s instructions.

BxPC3^luc cells were exposed to selected drugs for 48 h, all cells were harvested and the protein level of ABCG2 and ABCB1 was evaluated. The membrane protein was extracted according to the manufacturer’s instructions for the ProteoJET Membrane Protein Extraction Kit (Fermentas, Burlington, Ontario, Canada). The cell lysate (50 µg) was subjected to electrophoresis on 4–12% sodium dodecyl sulfate (SDS)–polyacrylamide gels followed by transfer onto a polyvinylidene difluoride (PVDF) membrane using a glycine transfer buffer. After blocking in a blocking buffer containing 5% nonfat milk for 1 hour at room temperature, the membrane was incubated with anti-ABCG2 and anti-ABCB1 primary antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) overnight at 4 ℃, and incubated with goat anti-rabbit IRDye 800CW secondary antibody (LICOR Inc., Lincoln, NE, USA) for 2 hours at room temperature. The β-actin antibody and Na⁺/K⁺ ATPase α (H-3) were served as a loading control for total protein and membrane protein expression assessments respectively. Then the bands were visualized by Odyssey infrared imaging system (LICOR Inc., Lincoln, NE, USA).

Skin tissues were subjected to total protein extraction using a ProteoJET Membrane Protein Extraction Kit (Fermentas), and then quantified using the Bradford assay. Equal amounts of soluble protein were separated by SDS/PAGE and transferred onto a polyvinylidene difluoride membrane (PVDF, Roche). Immunoblotting was conducted using antibodies specific for FGF5 (1:1000, Sigma-Aldrich) and anti-GAPDH (1:1000, Sigma-Aldrich). Primary antibodies were visualized using a fluorescence imager system (Sagecreation). Variations in sample loading were corrected by normalizing.

Possible FH binding protein(s) in the midgut membrane extract were detected by using the ligand blot analysis[11 (link)]. Briefly, membrane proteins were extracted by incubating the A. stephensi mosquito midguts in the membrane extraction buffer (containing the protein inhibitor cocktail) from the ProteoJET membrane protein extraction kit (Fermentas, K0321) for 2 h at 4°C with constant shaking. Extracted membrane proteins were recovered by centrifugation at 16,000g for 15 min at 4°C. A volume equivalent to 1 midgut per lane was then loaded onto 10% SDS-PAGE gel under non-reducing conditions. Resolved membrane proteins were transferred to nitrocellulose membranes and overlaid after a blocking step with 3% non-fat milk for 1h with either 20% HIS (a source of FH) or PBS (a negative control for FH binding) and incubated overnight at 4°C. After 5 washes in PBS-T, the membranes overlaid earlier with HIS were further incubated in either PBS (a negative control for binding of the secondary antibody) or with 2 μg/ml of the monoclonal anti-human FH antibody (131X). After a 1-hour incubation at RT and 5 washes in PBS-T, the membranes were incubated in a 1:10,000 dilution of goat anti-mouse IgG coupled to HRP (NFH822, Perkin Elmer life Sciences, Inc.). After a 1-hour incubation at RT and 5 washes in PBS-T, the blots were developed as described above.

We performed SPR analysis using a BIAcore 2000 SPR biosensor (GE Healthcare). Secreted re-eIF5A was immobilized on a sensor chip CM5 using the amine coupling method. Plasma membrane proteins, which were extracted from cardiac myocytes that had been subjected to hypoxia (60 min)/reoxygenation (30 min) using a ProteoJET Membrane Protein Extraction Kit (Thermo Fisher Scientific Inc.), were analyzed for their interaction with secreted re-eIF5A protein. The anti-eIF5A mAbs (YSP5-45-36 and YSPN2-74-18) and bovine serum albumin (BSA) were used as positive and negative controls, respectively.

Total LLC cellular protein lysates of EoE suspension culture were prepared as previously described [10 (link)]. ProteoJET™ Membrane Protein Extraction Kit (Thermo Inc., San Jose, CA, USA) was used to prepare cytosolic and membrane protein extracts according to manufacturer’s instruction. Protein samples were then subjected to SDS-PAGE, electrotransferation, and western immunoblotting (IB). The chemiluminescence images of IBs were developed with CyECL reagents (Cyrusbioscience, Taipei, Taiwan) according to the manufacturer’s instruction.

Membrane and cytosolic proteins from U-251 MG cells were obtained from 100-cm dishes at 90% confluence with ProteoJET Membrane Protein Extraction Kit (ThermoFisher Scientific) according to the manufacturer's instruction.