Immunoblot analysis was performed as previously described21 (link). Briefly, total proteins were extracted with RIPA buffer [50 mM Tris–HCl (pH 8.0), 0.15 M sodium chloride, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate, 1% NP-40] and both protease and phosphatase inhibitor cocktails (FUJIFILM Wako). The proteins were subjected to 10% SDS-PAGE and transferred to a PVDF membrane (ThermoFisher Scientific). The membranes were blocked with 3% skim milk followed by incubation with UCP1 antibody (MAB6158, R&D Systems, MN, USA), UCP2 antibodies (AF4739, R&D Systems) or β-Actin antibody (A5316, Sigma-Aldrich) at 4 °C overnight. The membranes were incubated with HRP-conjugated secondary antibody (Santa Cruz Biotechnology, CA, USA) for 1 h at room temperature. Immunoreactive bands were detected by Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore, Darmstadt, Germany). Each band intensity was quantified by densitometry using ImageJ software (National Institutes of Health, Bethesda, USA). For evaluation of protein stability, 10 µg/ml Cycloheximide was treated for the time indicated before harvest. Full-length western blots are shown in Supplementary Fig. S7 .
Protease and phosphatase inhibitor cocktail
Manufactured by Fujifilm
Sourced in Japan
Protease and phosphatase inhibitor cocktails are laboratory products designed to inhibit the activity of proteases and phosphatases during protein extraction and analysis. They help preserve the integrity of target proteins by preventing degradation and unwanted modifications. These cocktails contain a combination of specific inhibitors that target a wide range of proteases and phosphatases commonly found in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Hrp conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
β actin antibody a5316, by Merck Group (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
Ucp1 antibody mab6158, by R&D Systems (1 mentions)
Irdye 680 donkey anti rabbit igg, by LI COR (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Colorimetric assay kit, by Bio-Rad (1 mentions)
Ecl system, by GE Healthcare (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Ripa buffer, by Fujifilm (1 mentions)
Ripa buffer, by Nacalai Tesque (1 mentions)
Anti β actin gtx109639, by GeneTex (1 mentions)
Anti rabbit or anti mouse igg antibody cat 7074, by Cell Signaling Technology (1 mentions)
Cat 7076, by Cell Signaling Technology (1 mentions)
Mini protean tgx precast gel, by Bio-Rad (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Supersignal west femto trial kit, by Thermo Fisher Scientific (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Ab109616, by Abcam (1 mentions)
4 protocols using protease and phosphatase inhibitor cocktail
1
Immunoblot Analysis of UCP1 and UCP2 Expression
2
Investigating eNOS Regulation in PAR-2 KO Mice
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C48/80 (2 mg/kg) was administered intravenously to WT and PAR-2 KO mice, and the lungs were collected before administration and at 10 and 120 min after administration. The lungs were homogenized in RIPA buffer (50 mmol/L Tris-HCl [pH 7.4], 150 mmol/L NaCl, 5 mmol/L EDTA, 1% Triton X-100, 10 mmol/L NaF) containing protease and phosphatase inhibitor cocktails (Wako, Osaka, Japan). For the detection of eNOS and phosphorylated eNOS, the samples (25 μg per lane) were separated by electrophoresis and transferred to PVDF membranes.
The membranes were blocked with 5% BSA at room temperature for 30 min and incubated with anti-eNOS (clone: D9A5L, Cell Signaling Technology, MA, USA, 1:1,000) and anti-Ser177 p-eNOS (clone: C9C3, Cell Signaling Technology, 1:1,000) antibodies. Samples were also probed with an anti-β-actin antibody (GeneTex, CA, USA, 1:1,000) to normalize protein loading. The membrane was then incubated with secondary antibody (donkey anti-rabbit IgG IRDye 680, LICOR Biosciences, NE, USA, 1:10,000) at room temperature for 1 h. The bands were quantified using the Odyssey Imaging System (LICOR Biosciences).
The membranes were blocked with 5% BSA at room temperature for 30 min and incubated with anti-eNOS (clone: D9A5L, Cell Signaling Technology, MA, USA, 1:1,000) and anti-Ser177 p-eNOS (clone: C9C3, Cell Signaling Technology, 1:1,000) antibodies. Samples were also probed with an anti-β-actin antibody (GeneTex, CA, USA, 1:1,000) to normalize protein loading. The membrane was then incubated with secondary antibody (donkey anti-rabbit IgG IRDye 680, LICOR Biosciences, NE, USA, 1:10,000) at room temperature for 1 h. The bands were quantified using the Odyssey Imaging System (LICOR Biosciences).
3
Osteogenic Differentiation of iPSCs
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Osteogenic differentiation from human iPSCs of control and OI #3 was performed as mentioned above. MSCs were differentiated into osteoblasts as mentioned above for 2 weeks with or without 0.5 mM 4-PBA. Total proteins were extracted from induced osteoblasts in RIPA buffer (Wako, Tokyo, Japan) containing protease and phosphatase inhibitor cocktail (Wako, Tokyo, Japan). After centrifugation (15,000g, 15 min, 4 °C), supernatants were stored at −80 °C. Protein concentration was measured using a colorimetric assay kit (Bio-Rad, Hercules, CA). Proteins were subjected to SDS-PAGE and transferred to a PVDF membrane (Bio-Rad, Hercules, CA). After blocking, the membrane was incubated with primary antibody against alkaline phosphatase (R&D Systems, Minneapolis, MN #AF2910) diluted 1:200 and HRP-conjugated anti-β-actin antibody (MBL, Nagoya, Japan #PM053–7) diluted 1:5000. After incubation with the secondary antibody, the signals were visualized using ECL system (GE Healthcare, Bucks, UK). Densitometry was quantified with ImageJ 1.50i (National Institutes of Health, Bethesda, MD).
4
Western Blot Analysis of HIF-1α and HIF-2α
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Cells were lysed in RIPA Buffer (Nacalai Tesque, Inc., Kyoto, Japan) containing a protease and phosphatase inhibitor cocktail (Fujifilm Wako Pure Chemical Corporation, Kyoto, Japan). Protein concentration was determined using a Pierce BCA protein assay kit (Thermo Fisher Scientific). Ten micrograms of protein were denatured in Laemmli sample buffer and loaded on 4–15% Mini-PROTEAN TGX Precast Gels (Bio-Rad Laboratories, Richmond, CA, USA). After electrophoretic separation, the proteins were transferred onto PVDF membranes (MilliporeSigma, Billerica, MA, USA). Membranes were blocked overnight at 4 °C with Tris-buffered saline (TBS) containing 5% non-fat dried milk or bovine serum albumin. Following blocking, membranes were incubated overnight at 4 °C with primary antibodies, including anti-HIF-1α (ab179483; 1:1000; Abcam, Cambridge, UK), anti-HIF-2α (ab109616; 1:1000; Abcam), and anti-β-actin (GTX109639; 1:1000; GeneTex, Irvine, CA, USA). Proteins were incubated with secondary antibodies (anti-rabbit or anti-mouse IgG antibody; Cat. 7074 and Cat. 7076, respectively; 1:20,000; Cell Signaling Technology, Danvers, MA, USA) for 1 h at room temperature in TBS containing 5% non-fat dried milk. The chemiluminescence signal was detected using enhanced chemiluminescent substrate (SuperSignal™ West Femto Trial Kit; Thermo Fisher Scientific).
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