Cell were lysed in RIPA buffer supplemented with cOmplete® Protease Inhibitor Cocktail (Roche) and PhosSTOP® Phosphatase Inhibitor Cocktail (Roche). The lysates were centrifuged at 20,000g at 4 °C for 15 min, and the supernatants were incubated with DTT (100 mM) and NuPAGE® LDS Sample Buffer (Invitrogen) at 70 °C for 10 min. Proteins were separated on Novex® 4–12% Tris-Glycine Mini Gels (Invitrogen) and transferred to a PVDF membrane. The membrane was incubated with 5% skim milk at room temperature for 1 hr and subsequently with primary antibodies at 4 °C for overnight. For the detection of FABP7, the step of centrifuging cell lysates was omitted, and 5% BSA was used as a blocking solution. Primary antibodies were as follows and used at 1:1000 dilution unless otherwise stated: rabbit anti-FABP7 (#13347, Cell Signaling Technology), rabbit anti-UCP1 (U6382, Sigma Aldrich), rabbit anti-PGC-1α (1:200 v/v) (sc-13067, Santa Cruz Biotechnology), rabbit anti-PRDM16 (1:500 v/v) (ab106410, Abcam), rabbit anti-CREB (#9197, Cell Signaling Technology), and rabbit anti-phospho-CREB (#9198, Cell Signaling Technology). Appropriate secondary horseradish peroxidase-linked antibodies were used (Dako, UK). Immunoreactivity was detected with ECL Prime Western Blotting Detection Reagent (Amersham) and visualized using ImageQuant LAS 4000 mini (GE Healthcare).
Rabbit anti ucp1
Manufactured by Merck Group
Sourced in United States
Rabbit anti-UCP1 is a primary antibody that targets the uncoupling protein 1 (UCP1), which is a key regulator of thermogenesis in brown adipose tissue. This antibody can be used for the detection and analysis of UCP1 in various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Imagequant las 4000 mini, by GE Healthcare (1 mentions)
Novex 4 12 tris glycine mini gels, by Thermo Fisher Scientific (1 mentions)
Phosstop phosphatase inhibitor cocktail, by Roche (1 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Rabbit anti creb, by Cell Signaling Technology (1 mentions)
Ecl prime western blotting detection reagent, by Cytiva (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Rabbit anti fabp7, by Cell Signaling Technology (1 mentions)
Rabbit anti phospho creb, by Cell Signaling Technology (1 mentions)
Las 4000, by Cytiva (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Agilent Technologies (1 mentions)
Western blotting luminol reagent, by Santa Cruz Biotechnology (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Trans blot turbo system, by Bio-Rad (1 mentions)
Anti parp sc 7150, by Santa Cruz Biotechnology (1 mentions)
Anti actin, by Santa Cruz Biotechnology (1 mentions)
3 protocols using rabbit anti ucp1
1
Protein Expression Analysis in Adipocytes
2
Determination of UCP1 Protein in Adipose Tissue
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For UCP1 protein determination, mitochondrial fractions were extracted from approximately 50 mg of inguinal white and interscapular brown adipose tissue as previously described [29 (link)]. Briefly, adipose tissue samples were homogenized in 600 μl of supplemented sucrose buffer containing 250 mM sucrose, 20 mM HEPES (pH 7.4), 1mM EGTA, 1 mM DTT, 1 mM PMSF and protease inhibitor cocktail (Roche, Basel, Switzerland). The homogenates were then centrifuged at 500 x g for 12 min at 4°C in order to pellet the nucleus and cell debris. The supernatant was then centrifuged at 12000 x g for 20 min at 4°C and the supernatant removed. The remaining pellet was then resuspended in 100 μl of supplemented sucrose buffer and the protein concentration was determined by BCA assay (Thermo Scientific, MA, USA).
Proteins were separated by SDS-polyacrylamide gel electrophoresis and then transferred to a PVD membrane using a Trans-Blot Turbo System (Bio-Rad, CA, USA). The membranes were then probed with rabbit anti-UCP1 (1:750; Sigma-Aldrich, MO, USA), followed by washing, and probing with horseradish peroxidase-conjugated secondary antibody (1:5000; Dako, Glostrup, Denmark). Bands were visualized with Western Blotting Luminol Reagent (Santa Cruz, TX, USA) and imaged with the ChemiDoc MP Imaging System (Bio-Rad, CA, USA).
Proteins were separated by SDS-polyacrylamide gel electrophoresis and then transferred to a PVD membrane using a Trans-Blot Turbo System (Bio-Rad, CA, USA). The membranes were then probed with rabbit anti-UCP1 (1:750; Sigma-Aldrich, MO, USA), followed by washing, and probing with horseradish peroxidase-conjugated secondary antibody (1:5000; Dako, Glostrup, Denmark). Bands were visualized with Western Blotting Luminol Reagent (Santa Cruz, TX, USA) and imaged with the ChemiDoc MP Imaging System (Bio-Rad, CA, USA).
3
Subcellular Fractionation and Western Blotting
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Subcellular fractionation and Western blotting analysis was carried out as previously described [16] . Lysates were subjected to differential centrifugation to isolate the nuclear and mitochondrial fraction. Proteins were extracted by incubation in boiling sample buffer followed by sonication.
Fifty µg of total lysates or thirty µg of nuclear or mitochondrial fractions were separated by 10% SDS-PAGE and analysed by Western blotting. Mouse monoclonal antibodies anti-VDR (sc-13133), anti E-Cadherin (sc-21791) and goat antibody anti-UCP2 (sc-6525) were from Santa Cruz, CA, USA. Rabbit anti-UCP1 was obtained from Sigma (U6382). Our previous works [17, 18] have shown that the antibody against VDR gives some unspecific signal. The correct band was identified in past studies by molecular weight and silencing experiments in HaCaT, MCF7 and HeLa cells [17, 18] , and corresponds to the lower band when a doublet band is present. The signal that detects VDR is indicated in all figures. The loading controls were carried out on the same membranes and detected by antibodies anti-VDAC (monoclonal anti-porin 31HL, Calbiochem), anti-actin (mouse monoclonal sc-8432 Santa Cruz) and rabbit antibody anti-PARP (sc-7150, Santa Cruz).
Fifty µg of total lysates or thirty µg of nuclear or mitochondrial fractions were separated by 10% SDS-PAGE and analysed by Western blotting. Mouse monoclonal antibodies anti-VDR (sc-13133), anti E-Cadherin (sc-21791) and goat antibody anti-UCP2 (sc-6525) were from Santa Cruz, CA, USA. Rabbit anti-UCP1 was obtained from Sigma (U6382). Our previous works [17, 18] have shown that the antibody against VDR gives some unspecific signal. The correct band was identified in past studies by molecular weight and silencing experiments in HaCaT, MCF7 and HeLa cells [17, 18] , and corresponds to the lower band when a doublet band is present. The signal that detects VDR is indicated in all figures. The loading controls were carried out on the same membranes and detected by antibodies anti-VDAC (monoclonal anti-porin 31HL, Calbiochem), anti-actin (mouse monoclonal sc-8432 Santa Cruz) and rabbit antibody anti-PARP (sc-7150, Santa Cruz).
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