Western blot analysis was performed as described previously45 (link) using the following antibodies: anti-β-actin (Sigma), anti-α-tubulin (Calbiochem, Darmstadt, Germany), anti-IRF1 and anti-TNFR1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-lamin A/C (Novacastra, Berlin, Germany). Donkey anti-mouse IgG or donkey anti-rabbit IgG labeled with IRDye infrared dyes were used for fluorescence detection at 700 nm 800 nm (LI-COR Biotechnology, Bad Homburg, Germany). IRF1 was immunodetected by enhanced chemoluminescence (Amersham Biosciences, Freiburg, Germany) using an anti-mouse IgG-HRP as the secondary antibody. Cytosolic and nuclear extracts were prepared as described previously.46 (link) Briefly, for isolation of nuclear proteins, pelleted nuclei were resuspended in RIPA buffer, sonicated and nuclear supernatants were obtained by centrifugation.
Enhanced chemoluminescence
Manufactured by Cytiva
Sourced in Germany, United Kingdom
Enhanced chemoluminescence is a sensitive detection method used in molecular biology and biochemistry. It involves the generation of light through a chemical reaction, which is then detected and measured. This technique is commonly used for the identification and quantification of proteins in Western blot analysis.
Automatically generated - may contain errors
Lab products found in correlation
Immobilon p membrane, by Merck Group (2 mentions)
Donkey anti rabbit igg, by LI COR (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Donkey anti mouse igg, by LI COR (1 mentions)
Anti cx43, by Merck Group (1 mentions)
Apyrase, by Merck Group (1 mentions)
Small interfering rna sirna, by Santa Cruz Biotechnology (1 mentions)
Lipofectamine, by Merck Group (1 mentions)
Recombinant human tgf β1, by Merck Group (1 mentions)
Fibronectin, by R&D Systems (1 mentions)
Smad3, by Cell Signaling Technology (1 mentions)
Anti β actin antibody, by Merck Group (1 mentions)
4 protocols using enhanced chemoluminescence
1
Western Blot Analysis of Cellular Proteins
2
Western Blot Analysis of Protein Samples
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Tissues and cells were harvested and lysed in buffer (62.5 mmol/L Tris‐HCl, [pH 6.8], 10% glycerol, 2% SDS, 5% 2‐mercaptoethanol, and 0.5% bromphenol blue). Proteins were separated on 15% SDS‐polyacrylamide gels and electroblotted onto Immobilon‐P membranes (Millipore). Blots were incubated with a 1:1000 dilution of MAb 85.1 and detected by enhanced chemoluminescence according to the manufacturer's specifications (Amersham).
3
Proximal Tubule Epithelial Cell Culture
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Human primary proximal tubule epithelial cells (hPTECs) and human derived proximal tubule kidney (HK2) epithelial cells were both obtained from ATCC. Tissue culture supplies were purchased from Invitrogen (Paisley, UK). Immobilon P membrane was from Millipore (Watford, UK) and enhanced chemoluminescence from Amersham Biosciences (Amersham, UK). Antibodies Smad2 and Smad3 are monoclonal antibodies that were obtained from Cell signaling Technology ( Danvers, MA, USA). Small interfering RNA (siRNA) was obtained from Santa Cruz (Santa Cruz, CA, USA). For western blot analysis, CX26 and IL6 antibodies were obtained from Abcam (Cambridge, UK) and fibronectin from R&D Systems (Abingdon, UK), whilst CX43 antibody was obtained from Santa Cruz . For immunohistochemistry anti-CX26 was obtained from (Santa Cruz, CA, USA) and anti-CX43 (Sigma-Aldrich,MO, USA). Recombinant human TGF-β1, glucose, apyrase and lipofectamine were obtained from Sigma (Poole, UK), as were all other general chemicals. The anti-TGF-β1 ELISA was from R&D Systems. ATP biosensors were from Sarissa Biomedical Ltd (UK) and fluorodishes from WPI (Hertfordshire, UK).
4
Assessing Tumor and Endothelial Cell Proliferation
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The proliferative activity of tumor and endothelial cells was assessed with the colorimetric alamarBlue assay as described in detail in (15) . Four western blot analysis cells were incubated for 24 hours and one hour with patupilone and everolimus, respectively, followed by incubation under normoxic or hypoxic conditions (0.2% pO2; Invivo2 400 hypoxia workstation, Biotrace International, Bridgend, UK) and cell lysis in lysis buffer (63mM Tris, 10% Glycerol, 2% SDS, pH 6.8 + DTT) and SDS-PAGE. Antibody detection was achieved by enhanced chemoluminescence (Amersham, Piscataway, NJ) using a polyclonal rabbit anti-HIF-1α subunit antibody (Santa Cruz Biotechnology, #sc-10790, 1:1000), a rabbit polyclonal anti-phospho-P70-S6kinase antibody (Cell Signalling Technology, #9205, 1:1000), a rabbit polyclonal anti-P70-S6kinase antibody (Cell Signalling Technology, #9202, 1:1000) and a mouse monoclonal anti-β-actin antibody (Sigma Aldrich, #A5441, 1:1000). All experiments were carried out independently at least three times.
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