Cells were lysed in RIPA buffer supplemented with complete protease inhibitor cocktail (Roche, Mannheim, Germany). Nuclear and cytoplasmic fractions were performed using the ProteoJET Cytoplasmic and Nuclear Protein Extraction Kit (Fermentas, St. Leon-Rot, Germany). Immunoblots were performed as described previously [19 (link)]. Supernatant was harvested 24h and 48h after treatment. Immunoblots were probed with primary antibodies against SSTR1-5 (abcam, Cambridge, UK), PARP, AKT, pAKT, ERK, pERK, pRPS6, RPS6, eiF4E, PCNA, Cyclin D1, CDK4, chromogranin A and beta-actin (Sigma-Aldrich). Peroxidase-conjugated secondary antibodies against mouse or rabbit were obtained from Amersham (Freiburg, Germany) and goat from Sigma. Blots were detected by Western Lightning ECL (PerkinElmer, Rodgau, Germany).
Western lightning ecl
Manufactured by PerkinElmer
Sourced in United States
Western Lightning ECL is a chemiluminescent detection reagent used for the detection of proteins in Western blotting applications. It is designed to provide a sensitive and quantitative signal for the visualization of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Sigmafast protease inhibitor cocktail, by Merck Group (16 mentions)
α tubulin, by Merck Group (16 mentions)
Nitrocellulose membrane, by Bio-Rad (16 mentions)
Chemostar imager, by Intas (16 mentions)
Poinceau, by Merck Group (14 mentions)
Protease inhibitor cocktail, by Roche (4 mentions)
Phosstop, by Roche (4 mentions)
β actin, by Merck Group (4 mentions)
Immobilon p, by Merck Group (3 mentions)
Nkx2 2, by Aviva Systems Biology (2 mentions)
Smad1, by Santa Cruz Biotechnology (2 mentions)
Micro bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Erk sc 94, by Santa Cruz Biotechnology (2 mentions)
Hgt agarose, by Lonza (2 mentions)
Ponceau, by Merck Group (2 mentions)
Acrylamide gel, by Thermo Fisher Scientific (2 mentions)
Iblot system, by Thermo Fisher Scientific (2 mentions)
Phospho akt, by Cell Signaling Technology (2 mentions)
Proteojet cytoplasmic and nuclear protein extraction kit, by Thermo Fisher Scientific (1 mentions)
49 protocols using western lightning ecl
1
Cellular Fractionation and Immunoblot Analysis
2
Standardized Western Blotting Protocol
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Western blots were generated by the semi-dry method. Protein lysates from cell lines were prepared using SIGMAFast protease inhibitor cocktail (Sigma, Taufkirchen, Germany). Proteins were transferred onto nitrocellulose membranes (Bio-Rad, München, Germany) and blocked with 5% dry milk powder dissolved in PBS. The following antibodies were used: alpha-Tubulin (Sigma, Taufkirchen, Germany), HOXB5 (Santa Cruz Biotechnology, Heidelberg, Germany), and BCL6 (Cell Signaling Technology, Danvers, MA, USA). For loading control, blots were reversibly stained with Poinceau (Sigma, Taufkirchen, Germany) and detection of alpha-Tubulin (TUBA) performed thereafter. Secondary antibodies were linked to peroxidase for detection by Western Lightning ECL (Perkin Elmer, Waltham, MA, USA). Documentation was performed using the digital system ChemoStar Imager (INTAS, Göttingen, Germany).
3
Western Blot Analysis of CCND1 Protein
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Samples were lysed in 50 mM Tris-HCl (Sigma Aldrich, St. Louis, MO, USA, Cat# T2663), pH 7.4, 1% NP-40 (Sigma Aldrich, Cat# I8896), 150 mM NaCl (Sigma Aldrich, Cat# S5886), 1 mM EDTA (Promega, Madison, WI, USA, Cat# V4231), 1% glycerol (Sigma Aldrich, Cat# G5516), 100 μM vanadate (Sigma Aldrich, Cat# S6508), protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland, Cat# 004693159001), and PhosSTOP (Roche Diagnostics, Cat# 04906837001). Lysates were subjected to SDS-PAGE and transferred to PVDF membranes. The proteins were detected by western blot analysis by using an enhanced chemiluminescence system (Western Lightning–ECL, PerkinElmer, Waltham, MA, USA, Cat# NEL103001EA). Antibodies used were specific for CCND1 1:1000 (Cell Signaling, Danvers, MA, USA, Cat# 2926), and GAPDH 1:5000 (Abcam, Cambridge, UK, Cat# ab9485). All western blot quantifications were performed using ImageJ72 (link).
4
Western Blot Analysis of Signaling Proteins
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Western blots were generated by the semi-dry method. Protein lysates from cell lines were prepared using SIGMAFast protease inhibitor cocktail (Sigma). Proteins were transferred onto nitrocellulose membranes (Bio-Rad, München, Germany) and blocked with 5% dry milk powder dissolved in phosphate-buffered-saline buffer (PBS). The following antibodies were used: MSX1 (R & D Systems), alpha-Tubulin (Sigma), PDGFRB (R & D Systems), phospho-PDGFRB (Aviva Systems Biology, Eching, Germany), NKX2-2 (Aviva Systems Biology) and PITX1 (Abnova, Taipei, Taiwan). For loading control blots were reversibly stained with Poinceau (Sigma) and detection of alpha-Tubulin (TUBA) was performed thereafter. Secondary antibodies were linked to peroxidase for detection by Western-Lightning-ECL (Perkin Elmer, Waltham, MA, USA). Documentation was performed using the digital system ChemoStar Imager (INTAS, Göttingen, Germany). PDGFD and BMP4 were quantified in the medium by ELISA using according Quantikine ELISA kits from R & D Systems. Samples were obtained by harvesting supernatants of 1x106 cells which were washed in PBS and subsequently incubated in 1 ml medium for 24 h.
5
Western Blot Analysis of IRF4 Expression
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Western blots were generated by the semi-dry method. Protein lysates from cell lines were prepared using SIGMAFast protease inhibitor cocktail (Sigma). Proteins were transferred onto nitrocellulose membranes (Bio-Rad, München, Germany) and blocked with 5% dry milk powder dissolved in phosphate-buffered-saline buffer (PBS). The following antibodies were used: alpha-Tubulin (Sigma) and IRF4 (NOVUS Biologicals, Colorado, USA). For loading control blots were reversibly stained with Poinceau (Sigma) and detection of alpha-Tubulin (TUBA) was performed thereafter. Secondary antibodies were linked to peroxidase for detection by Western-Lightning-ECL (Perkin Elmer, Waltham, MA, USA). Documentation was performed using the digital system ChemoStar Imager (INTAS, Göttingen, Germany).
6
Immunoprecipitation and Western Blot
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Cells were rinsed in ice-cold PBS and lysed in lysis buffer (20 mM Hepes, pH 7.2, 1% NP-40 substitute, and 150 mM NaCl supplemented with the protease inhibitors benzamidine, PMSF, aprotinin, and leupeptin) on ice. Lysates were clarified by centrifugation and protein concentration was determined by BCA assay (Thermo Fisher Scientific). Lysates were diluted to 1 to 2 mg/ml and incubated with the indicated primary antibody overnight at 4°C. The next day, samples were incubated with protein G–Sepharose (GE Healthcare) for 4 h. Immunoprecipitates were washed five times in lysis buffer and eluted by incubating at 90°C. Samples were separated by SDS-PAGE and transferred to nitrocellulose membrane. Membranes were blocked in 5% milk in PBS plus 0.1% Tween-20 (PBST), and primary antibodies were diluted in blocking buffer or 3% BSA/PBST. Primary antibody incubations were performed overnight at 4°C. The following day, membranes were washed with PBST, blocked again as before, and incubated with HRP-conjugated secondary antibodies, diluted in 5% milk/PBST, for one hour at room temperature. Membranes were then washed with PBST before protein detection with Western Lightning ECL and Plus-ECL kits (PerkinElmer) according to the manufacturer’s instructions.
7
Protein expression analysis by Western blot
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Western blots were generated by the semi-dry method. Proteins obtained from cell line lysates using SIGMAFast protease inhibitor cocktail (Sigma) were transferred onto nitrocellulose membranes (Bio-Rad, München, Germany) and blocked with 5% dry milk powder dissolved in phosphate-buffered-saline buffer (PBS). The following antibodies were used: alpha-Tubulin (Sigma), AUTS2 (Origene), SMAD1 (Santa Cruz Biotechnology, Heidelberg, Germany), STAT5 (Santa Cruz Biotechnology), phospho-STAT5 (Cell Signaling Technology, Danvers, MA, USA). For loading control the blots were reversibly stained with Poinceau (Sigma) and then detection of alpha-Tubulin (TUBA) or SMAD1 was performed. Secondary antibodies were linked to peroxidase for detection by Western-Lightning-ECL (Perkin Elmer, Waltham, MA, USA). Documentation was performed using the digital system ChemoStar Imager (INTAS, Göttingen, Germany).
8
Western Blot Analysis and FGF2 ELISA
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Western blots were generated by the semi-dry method. Proteins obtained from cell line lysates using SIGMAFast protease inhibitor cocktail (Sigma) were transferred onto nitrocellulose membranes (Bio-Rad, München, Germany) and blocked with 5% dry milk powder dissolved in phosphate-buffered-saline buffer (PBS). The following antibodies were used: alpha-Tubulin (Sigma), OTX1 (Santa Cruz Biotechnology, Heidelberg, Germany), OTX2 (Abcam, Cambridge, UK), and ZHX1 (Santa Cruz Biotechnology). For loading control the blots were stained with Poinceau (Sigma) and then detection of alpha-Tubulin (TUBA) was performed. Secondary antibodies were linked to peroxidase for detection by Western-Lightning-ECL (Perkin Elmer, Waltham, MA, USA). Documentation was performed using the digital system ChemoStar Imager (INTAS, Göttingen, Germany).
Quantification of FGF2 protein in cell culture supernatants was performed as follows: log-phase cells were washed twice in PBS and cultivated in fresh medium at 1x106 cells per ml and supernatants harvested after 24 h. ELISA was performed using the Human FGF basic Quantikine ELISA Kit (R&D Systems) and an ELISA reader (Thermo Electron, Vantaa, Finland).
Quantification of FGF2 protein in cell culture supernatants was performed as follows: log-phase cells were washed twice in PBS and cultivated in fresh medium at 1x106 cells per ml and supernatants harvested after 24 h. ELISA was performed using the Human FGF basic Quantikine ELISA Kit (R&D Systems) and an ELISA reader (Thermo Electron, Vantaa, Finland).
9
Adipogenic Differentiation Assay
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Dexamethasone (DXM), bovine pancreatic insulin, 3-isobutyl-1-methylxanthine (IBMX), dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich. Rosiglitazone came from Alexis Biochemicals (Hornby, ON). Dulbecco’s Modified Eagle Medium was from Wisent Inc. (St-Bruno, QC). For the measurement of triglyceride content, the AdipoRed reagent was used (Lonza Walkersville Inc., Walkersville, MD). We also used the Western Lightning ECL from Perkin Elmer (Waltham, MA). Aminoimidazole carboxamide ribonucleotide (AICAR) was purchased from Toronto Research Chemicals (Toronto, ON). The measurement of protein was carried out with an assay kit from Bio-Rad (Mississauga, ON).
10
Western Blot Protein Analysis
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