Whole-cell lysates were prepared using PRO-PREP™ protein extraction solution (iNtRON Biotechnology, Seongnam, Korea) in the presence of a phosphatase inhibitor cocktail (Set V; Calbiochem, Darmstadt, Germany). The protein content of each cell lysate was quantified using the Bio-Rad Protein Assay system (Bio-Rad Laboratories), then lysates containing equivalent quantities of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes (EMD Millipore Corporation, MA, USA). Membranes were blocked for 1 h at room temperature with 5% nonfat dry milk (BD Biosciences, NJ, USA) in TBS-T (TBS containing 0.05% Tween 20) and hybridized with the indicated primary antibodies overnight at 4°C. Membranes were then washed three times with TBS-T and incubated with peroxidase-conjugated goat anti-mouse or anti-rabbit secondary antibodies for 1 h at room temperature. Immunoreactive bands were visualized using Clarity™ Western ECL Blotting Substrate (Bio-Rad Laboratories), followed by exposure to X-ray film (CP-BU new; AGFA, Mortsel, Belgium).
Cp bu new
Manufactured by AGFA HealthCare
Sourced in Belgium
The CP-BU new is a compact medical imaging processor designed for use in healthcare facilities. It is capable of processing and developing various types of medical films and plates. The core function of the CP-BU new is to provide reliable and efficient image processing for diagnostic imaging needs.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by GE Healthcare (3 mentions)
Curix 60 developer, by AGFA HealthCare (2 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Nonfat dry milk, by BD (1 mentions)
Protein assay system, by Bio-Rad (1 mentions)
Pro prep protein extraction solution, by iNtRON Biotechnology (1 mentions)
Clarity western ecl blotting substrate, by Bio-Rad (1 mentions)
P8340, by Merck Group (1 mentions)
Protran, by Cytiva (1 mentions)
Bradford reagent, by Bio-Rad (1 mentions)
Protease phosphatase inhibitor cocktail, by Roche (1 mentions)
Ab10983, by Abcam (1 mentions)
Ag 20b 0014 c100, by Adipogen (1 mentions)
Ag 20b 0042 c100, by Adipogen (1 mentions)
Anti dig antibody, by Roche (1 mentions)
Bamhi, by New England Biolabs (1 mentions)
Bamhi, by Roche (1 mentions)
Biodrop duo, by Harvard Bioscience (1 mentions)
Hydrogen peroxide, by Merck Group (1 mentions)
Luminol, by Merck Group (1 mentions)
8 protocols using cp bu new
1
Western Blot Protein Quantification
2
Protein Extraction and Western Blot Analysis
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The cells were lysed on ice for 10 min with RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycolate, 0.1% SDS, 50 mM Tris-HCl at pH 8.0, 10 mM NaF, 1 mM Na3VO4, 1 mM EDTA, and 1 mM EGTA) containing a protease inhibitor cocktail (P8340; Sigma-Aldrich) and centrifuged with 12,000 rpm for 10 min at 4°C. The supernatants were mixed with 4×SDS sample buffer (250 mM Tris-HCl at pH 6.8, 8% SDS, 40% glycerol, and 0.04% bromophenol blue) and 10 mM DTT (0281-25G; Amresco). Mixtures were boiled for 5 min. The protein samples were loaded in SDS polyacrylamide gels (3% stacking gel and 4–10% separating gel), electrophoresed, and transferred to Protran BA85 nitrocellulose membranes (10401196; GE Healthcare Life Sciences). The membranes were blocked with blocking solution (5% nonfat milk in 0.1% Tween 20 in TBS or 5% bovine serum albumin in 0.1% Tween 20 in TBS) for 2 h, incubated with primary antibodies diluted in blocking solution for 16 h at 4°C, washed four times with TBST (0.1% Tween 20 in TBS), incubated with secondary antibodies in blocking solution for 30 min, and washed again. To detect the signals of secondary antibodies, the ECL reagent (ABfrontier, LF-QC0101) and x-ray films (Agfa, CP-BU NEW) were used.
3
Quantitative Western Blot Analysis
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All protein fractions were assayed using the Bradford reagent (Bio-Rad) and then boiled in Laemmli sample buffer, resolved by SDS–PAGE, transferred onto a nitrocellulose membrane (GE Healthcare) and analysed by western blotting. Dot-blot filter retardation assays were performed in a 96-well BioDot microfiltration unit (Bio-Rad) using a 0.22 µm cellulose acetate membrane (Dutscher). After treatment, the samples were resuspended in 2% SDS, loaded onto the membrane, filtered and washed twice with 0.1% SDS. The list of primary antibodies is in Supplementary file 6 . Secondary horseradish-peroxidase-conjugated antibodies (anti-mouse and anti-rabbit) were purchased from Jackson ImmunoResearch Laboratories; anti-rat antibodies were purchased from Bethyl Laboratories. The membranes were incubated with commercially available (Pierce) or homemade enhanced chemiluminescence substrate as described in (Bertolin et al., 2016 (link)). Chemiluminescence signals were captured on film (CP-BU new, Agfa Healthcare), developed using CURIX 60 developer (Agfa Healthcare) and quantified with ImageJ software (NIH). The relative abundance of specific bands of interest was calculated by normalising it towards the abundance of loading controls and indicated in each graph.
4
Western Blot Analysis of BAT Proteins
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BAT and primary brown adipocytes were lysed using tissue extraction reagent I (Invitrogen, FNN0071) or NP40 cell lysis buffer (Invitrogen, FNN0021) containing protease/phosphatase inhibitor cocktail (Roche, 04693132001) and 1 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich, P7626). Soluble proteins (10 μg per lane) were separated on a 12% SDS polyacrylamide gel and blotted on a nitrocellulose membrane (GE Healthcare, 1060004). Membranes were incubated with primary antibody at 4°C overnight, and horseradish peroxidase-conjugated secondary antibody at RT for 1 h in 5% skim milk (Carl Roth, T145.2) with TBST, visualized using an chemiluminescence detection system (NLRP3: GeneDepot, W3680-010; CASP1: Biomax, BWD0100; UCP1 and ACTB: Biomax, BWP0200), and exposed to film (Agfa, CP-BU NEW). To determine the available linear range of western blots, the exposure time was collected in every experiment.
Anti-UCP1 (1:10,000 dilution) antibody was purchased from Abcam (ab10983). Anti-NLRP3 (1:1,000 dilution) and anti-CASP1 (1:2,000 dilution) antibodies were purchased from Adipogen (AG-20B-0014-C100, AG-20B-0042-C100). Anti-ACTB (1:20,000 dilution) antibody was purchased from Sigma-Aldrich (A5441). The signal intensities of protein bands were quantified with the ImageJ software (NIH) and normalized using the intensity of the loading control ACTB.
Anti-UCP1 (1:10,000 dilution) antibody was purchased from Abcam (ab10983). Anti-NLRP3 (1:1,000 dilution) and anti-CASP1 (1:2,000 dilution) antibodies were purchased from Adipogen (AG-20B-0014-C100, AG-20B-0042-C100). Anti-ACTB (1:20,000 dilution) antibody was purchased from Sigma-Aldrich (A5441). The signal intensities of protein bands were quantified with the ImageJ software (NIH) and normalized using the intensity of the loading control ACTB.
5
Genomic DNA Extraction and Southern Blot Analysis
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To extract genomic DNA, Xcg cells grown on TSA medium were subcultured into TSB medium (casein peptone 17 g, soya peptone 3 g, sodium chloride 5 g, dipotassium hydrogen phosphate 2.5 g, glucose 2.5 g in 1 liter distilled water) in a 28°C shaking incubator for 24 h. Bacterial DNA was extracted from 1 ml of cells grown to an optical density at 600 nm (OD600) of 0.8 to 1.0 using a commercial genomic DNA extraction kit (iNtRON Biotechnology, Inc., Seongnam, Korea) and the protocol for Gram-negative bacteria. The concentration of extracted DNA was estimated using a spectrophotometer (BioDrop Duo, Biochrom Ltd., Cambridge, UK).
For Southern blot analysis, genomic DNA of Xcg was digested with the restriction enzyme BamHI (New England Biolabs, Ipswich, MA, USA). Electrophoretic separation and transfer to nylon membranes were performed as described (Sambrook et al., 1989 ). For DNA hybridization, a digoxigenin (DIG)-labeled avrBs3 gene probe was used; this probe was comprised of a 3.3-kb internal BamHI fragment of avrBs3 cloned into pBlueScript, as described previously (Park et al., 2008 (link)), using a commercial protocol for DIG-labeled probes and membrane hybridization (Roche Diagnostics, Mortsel, Belgium). Detection of hybridization was performed using an anti-DIG antibody (Roche Diagnostics) and subsequent exposure to an X-ray film (Agfa CP-BU new).
For Southern blot analysis, genomic DNA of Xcg was digested with the restriction enzyme BamHI (New England Biolabs, Ipswich, MA, USA). Electrophoretic separation and transfer to nylon membranes were performed as described (Sambrook et al., 1989 ). For DNA hybridization, a digoxigenin (DIG)-labeled avrBs3 gene probe was used; this probe was comprised of a 3.3-kb internal BamHI fragment of avrBs3 cloned into pBlueScript, as described previously (Park et al., 2008 (link)), using a commercial protocol for DIG-labeled probes and membrane hybridization (Roche Diagnostics, Mortsel, Belgium). Detection of hybridization was performed using an anti-DIG antibody (Roche Diagnostics) and subsequent exposure to an X-ray film (Agfa CP-BU new).
6
Western Blot Analysis of Kinase Activation
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Samples obtained from kinase tests or total protein fractions were boiled in Laemli sample buffer, resolved by SDS–PAGE, transferred onto a nitrocellulose membrane (GE Healthcare) and analysed by western blotting. The following primary antibodies were used: monoclonal mouse anti-AURKA clone 35C1, 1:20 (ref. 68 (link)) or rabbit anti-AURKA pThr288 1:2,000 (Thermo Fisher Scientific, MA5-14904); and anti-Histone H3 pSer10, 1:10,000 (Merck Millipore, 06-570) or rat anti-TUBA1A clone YL1/2, 1:5,000 (EMD Millipore, MAB1864). Secondary horseradish peroxidase-conjugated antibodies anti-mouse or -rabbit were purchased from Jackson ImmunoResearch Laboratories (315-035-045 and 111-035-144), while anti-rat were purchased from Bethyl Laboratories (A110-105P). Membranes were incubated with commercially available (Pierce) or home-made enhanced chemiluminescence substrate composed of 100 mM Tris (pH 8.6), 13 mg ml−1 coumaric acid (Sigma), 44 mg ml−1 luminol (Sigma) and 3% hydrogen peroxide (Sigma). Chemiluminescence signals were captured on a film (CP-BU new, Agfa Healthcare) with a CURIX 60 developer (Agfa Healthcare). Uncropped scans of western blots are shown in Supplementary Fig. 5 .
7
Western Blot Analysis of WT1 Protein
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Proteins from the fresh frozen sarcoma tissue were loaded onto each well of the gel, separated by SDS-PAGE, and then transferred onto a membrane (CP-BU new, Agfa). After blocking nonspecific binding, the membrane was immunoblotted with the anti-WT1 mouse monoclonal antibody WLM 04 (Santa Cruz Biotechnology), followed by incubation with the appropriate secondary antibody conjugation with alkaline phosphatase.
8
Western Blot Protein Quantification
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Protein extracts from cultured cells or cell culture supernatants were quantified and electrophoresed as previously described [22 (link)]. Western blotting was performed using the appropriated dilutions of primary and secondary antibodies (usually 1:2000) (Table 1 ). Detection of α-tubulin with a specific antibody (Sigma) was used as a protein-loading control.
Colorimetric determinations to quantify total protein amounts in cell extracts and culture supernatants were performed with a Plate Reader Axis UVM340 (Biochrom). Western blotting images were obtained by developing exposed films (CP-BU New, Agfa) for 10–30 s (α-tubulin and HA fusion proteins), 1 min (DLK1, DLK2, NOTCH1, and NOTCH2 proteins), or 5 min (NOTCH3 and NOTCH4 proteins) with the Pierce ECL Plus Western blotting substrate kit (Thermo Scientific) in a Curix 60 developing apparatus (AGFA). Films were scanned with an HP Officejet Pro 8600 scanner and signals of the different proteins were quantified using the QuantityOne 4.6.5. (Basic) software.
Colorimetric determinations to quantify total protein amounts in cell extracts and culture supernatants were performed with a Plate Reader Axis UVM340 (Biochrom). Western blotting images were obtained by developing exposed films (CP-BU New, Agfa) for 10–30 s (α-tubulin and HA fusion proteins), 1 min (DLK1, DLK2, NOTCH1, and NOTCH2 proteins), or 5 min (NOTCH3 and NOTCH4 proteins) with the Pierce ECL Plus Western blotting substrate kit (Thermo Scientific) in a Curix 60 developing apparatus (AGFA). Films were scanned with an HP Officejet Pro 8600 scanner and signals of the different proteins were quantified using the QuantityOne 4.6.5. (Basic) software.
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