Sigma fast bcip nbt reagent

The proteins were transferred to nitrocellulose membranes and then pretreated for 2 h with Tris-buffered saline (TBS) containing 0.05 % Tween 20 (TBS-T) and 5 % non-fat dry milk, at room temperature. Membranes were probed for 16 h with a mixture containing monoclonal (mouse) anti-human ORP150 antibody (1: 100) or monoclonal (mouse) anti-human HIF-1α (1: 500) in 5 % dried milk in TBS-T, at 4 °C. Then the alkaline phosphatase conjugated antibody against mouse IgG (whole molecule) was added at a 1:2,500 dilution in TBS-T for 1 h with slow shaking. The nitrocellulose was washed with TBS-T (five times for 5 min) and exposed to Sigma-Fast BCIP/NBT reagent.

The proteins were transferred to nitrocellulose membranes and then pretreated for 2 h with Tris-buffered saline (TBS) containing 0.05% Tween 20 (TBS-T) and 5% nonfat dry milk, at room temperature. Membranes were probed with a mixture containing anti-ORP150 antibody (1 : 100) or anti-HIF-1α (1 : 500) in 5% dried milk in TBS-T, at 4°C, for 16 h. Then the alkaline phosphatase conjugated antibody against mouse IgG (whole molecule) was added at concentration 1 : 2500 in TBS-T for 1 h with slow shaking. The nitrocellulose was washed with TBS-T (five times for 5 min) and exposed to Sigma-Fast BCIP/NBT reagent.

Cells were washed with cold PBS and solubilized in 100 μL per well of passive lysis buffer. The lysates from each well were centrifuged at 10,000× g, at 4 °C, for 10 min. Samples of lysates containing 20 μg of protein were subjected to SDS-PAGE, as described by Laemmli [35 (link)]. The 12% polyacrylamide gel and constant current (25 mA) were used. The proteins were transferred to nitrocellulose membranes and subsequently pre-treated with Tris-buffered saline (TBS) containing 0.05% Tween 20 (TBS-T) and 5% non-fat dry milk at room temperature, for 2 h. Membranes were probed with the primary polyclonal (rabbit) anti-human LC3 I/II antibody (1:1000) in 5% dry milk in TBS-T at 4 °C, for 16 h. Subsequently, the alkaline phosphatase-conjugated secondary antibody against rabbit IgG (whole molecule) at 1:2500 dilution was added in TBS-T with slow shaking for 1 h. The membranes were washed with TBS-T and exposed to Sigma-Fast BCIP/NBT reagent (Sigma, St. Louis, MO, USA).

The Dulbecco’s modified Eagle’s medium (DMEM), containing glucose at 4.5 mg/mL with GlutaMax^TM, trypsin-EDTA, penicillin, streptomycin and fetal bovine serum Gold (FBS Gold) were provided by Gibco (San Diego, CA, USA). Passive lysis buffer, ReliaPrep RNA Cell Miniprep System and luminescent Caspase-Glo 9 Assay were provided by Promega (Madison, WI, USA), and BCA Protein Assay Kit by Thermo Scientific (Rockford, IL, USA). Annexin V Apoptosis Detection Kit I, JC-1 MitoScreen Kit, APO-Direct Kit were product of BD Pharmingen^TM (San Diego, CA, USA). Sigma-Fast BCIP/NBT reagent, acridine orange, fumed silica dioxide amorphous powder 7 nm, silica dioxide spherical, porous nanopowder 5–15 nm and silica dioxide nanopowder 10–20 nm, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and dichlorodihydrofluorescein diacetate were provided by Sigma (St. Louis, MO, USA). Polyclonal (rabbit) anti-human LC3 antibody and alkaline phosphatase-labeled anti-rabbit immunoglobulin G were provided by Cell Signaling Technology (Boston, MA, USA). High Capacity RNA-to-cDNA Kit was purchased from Applied Biosystems (Foster City, CA, USA).

Immunoblotting was performed essentially as described before (Knoller et al., 1991 (link)), with a number of modifications. The blocking buffer consisted of NaCl (136 mM), Tris-HCl (25 mM), and KCl (2.68 mM), pH 7.5, supplemented 3 g% of bovine serum albumin (A4053, 96%, Sigma-Aldrich) and 0.1% (v/v) Triton X-100. A Pierce G2 Fast Blotter semi-dry transfer apparatus and 1-Step Transfer Buffer (Prod. No. 84731) were used, following the manufacturer's instructions (Thermo Fisher Scientific). Second antibodies were alkaline phosphatase conjugates and bands were detected by direct color development, using SIGMAFAST BCIP-NBT reagent (B5655, Sigma-Aldrich).

Dulbecco’s modified Eagle’s medium (DMEM) containing glucose at 4.5 mg/ml (25 mM) with Glutamax, penicillin, streptomycin and trypsin–EDTA were provided by Invitrogen (San Diego, USA), passive lysis buffer by Promega (Madison, USA), FBS Gold by Gibco (San Diego, USA), BCA Protein Assay Kit by Thermo Scientific (Rockford, USA), PE Annexin V Apoptosis Detection Kit I by BD Pharmingen™ (CA, USA), Senescence Detection Kit by bioVision (CA, USA), Sigma-Fast BCIP/NBT reagent by Sigma (St Louis, MO, USA), monoclonal (mouse) anti-human ORP150 antibody by IBL (Gunma, Japan), monoclonal (mouse) anti-HIF-1α antibody by BD Biosciences (CA, USA), and alkaline phosphatase-labelled anti-mouse immunoglobulin G by Rockland (PA, USA), precision plus protein standards by Bio-Rad Laboratories (USA).