Experiments were performed as described1 (link). Fat tissues were grounded in RIPA buffer [50 mM Tris pH 7.5, 1 % NP40, 0.5 % sodium deoxycholate, 0.1 % SDS, 150 mM NaCl, 5 mM EDTA, and protease inhibitor cocktail (45 μg/ml, Roche, 11 873 580 001)] at 4 °C. Homogenates were separated in 10 % polyacrylamide gels and blotted to Hybond nitrocellulose membranes (GE Healthcare). Membranes were decorated using following antibodies: anti-LSD11 (link) (1/1000), anti-Prdm16 (abcam, ab118573, 1/500), anti-Ucp1 (abcam, ab10983, 1/1000), Fabp4 (Santa-Cruz, sc-18661, 1/2000), anti-Nrf1 (abcam, ab55744, 1/1000), anti-Flag (Sigma, F3165, 1/500), anti-β-Tubulin (Sigma, T6074, 1/10000), or anti-β-Actin (Sigma, A1978, 1/10000). Secondary antibodies conjugated to horseradish peroxidase (GE Healthcare) were detected using an enhanced chemiluminescence detection system (GE Healthcare). Protein levels were quantified using the Chemi Capt software (Peqlab). Uncropped scans are available in Supplementary Fig. 16 .
Chemi capt software
Manufactured by Avantor
Chemi Capt is a software application designed for data acquisition and analysis of chemiluminescence-based assays. It provides a user-friendly interface for setting up and running experiments, as well as tools for visualizing and interpreting the resulting data.
Automatically generated - may contain errors
Lab products found in correlation
Ab10983, by Santa Cruz Biotechnology (2 mentions)
Ab118573, by Abcam (2 mentions)
Ab55744, by Merck Group (2 mentions)
Horseradish peroxidase, by GE Healthcare (2 mentions)
Enhanced chemiluminescence detection system, by GE Healthcare (2 mentions)
A1978, by Merck Group (2 mentions)
Hybond nitrocellulose membrane, by GE Healthcare (2 mentions)
Sc 18661, by Abcam (2 mentions)
T6074, by Merck Group (2 mentions)
F3165, by Merck Group (2 mentions)
Anti β actin, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Ace glow, by Avantor (1 mentions)
Sc 25778, by Santa Cruz Biotechnology (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
3 protocols using chemi capt software
1
Immunoblotting Analysis of Adipose Tissue
2
Immunoblotting Analysis of Adipose Tissue
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Experiments were performed as described1 (link). Fat tissues were grounded in RIPA buffer [50 mM Tris pH 7.5, 1 % NP40, 0.5 % sodium deoxycholate, 0.1 % SDS, 150 mM NaCl, 5 mM EDTA, and protease inhibitor cocktail (45 μg/ml, Roche, 11 873 580 001)] at 4 °C. Homogenates were separated in 10 % polyacrylamide gels and blotted to Hybond nitrocellulose membranes (GE Healthcare). Membranes were decorated using following antibodies: anti-LSD11 (link) (1/1000), anti-Prdm16 (abcam, ab118573, 1/500), anti-Ucp1 (abcam, ab10983, 1/1000), Fabp4 (Santa-Cruz, sc-18661, 1/2000), anti-Nrf1 (abcam, ab55744, 1/1000), anti-Flag (Sigma, F3165, 1/500), anti-β-Tubulin (Sigma, T6074, 1/10000), or anti-β-Actin (Sigma, A1978, 1/10000). Secondary antibodies conjugated to horseradish peroxidase (GE Healthcare) were detected using an enhanced chemiluminescence detection system (GE Healthcare). Protein levels were quantified using the Chemi Capt software (Peqlab). Uncropped scans are available in Supplementary Fig. 16 .
3
Analyzing K1-5 Expression In Vitro and In Vivo
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To determine K1-5 expression in vitro, cells were transfected as described above. Two days later the cells were harvested in 100 μL PBS with protease inhibitors (Complete, Roche Diagnostics, Mannheim, Germany). For analysis of K1-5 expression in vivo, liver and tumour tissues (50 mm3) were harvested after termination of the in vivo experiments. Tumour and liver tissues were homogenised in 500 μL PBS and protease inhibitors (Complete) using the Precellys system (Peqlab, Heidelberg, Germany). Cells and tissue homogenisates were then lysed by repeated freeze-thaw cycles. Protein concentrations of tissue and cells were determined using the DC protein assay (Biorad, Munich, Germany) according to the manufacturer's protocol.
50 μg protein was used for SDS-polyacrylamide gel electrophoresis. Proteins were transferred on PVDF membrane (Biorad). Detection of K1-5 was done using an anti-angiostatin antibody (A1101, Sigma-Aldrich, Hamburg, Germany) and a corresponding secondary antibody (rabbit anti-goat, Santa Cruz, Heidelberg, Germany). GAPDH was used as a control (sc-25778, Santa Cruz). Chemiluminescent signal was developed using AceGlow (Peqlab) and the Chemismart system with ChemiCapt software (Peqlab).
50 μg protein was used for SDS-polyacrylamide gel electrophoresis. Proteins were transferred on PVDF membrane (Biorad). Detection of K1-5 was done using an anti-angiostatin antibody (A1101, Sigma-Aldrich, Hamburg, Germany) and a corresponding secondary antibody (rabbit anti-goat, Santa Cruz, Heidelberg, Germany). GAPDH was used as a control (sc-25778, Santa Cruz). Chemiluminescent signal was developed using AceGlow (Peqlab) and the Chemismart system with ChemiCapt software (Peqlab).
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