The detailed procedure was outlined in the previous study 21 . Briefly, cells were lysed using Lysis Buffer (KeyGEN BioTECH, Nanjing, China). Protein concentration was measured using a BCA Protein Assay Kit (KeyGEN BioTECH). Twenty micrograms of protein extract was separated by 10% SDS/PAGE, then transferred to nitrocellulose membrane (Promega, Madison, WI, USA) and incubated with the primary antibodies against SLC34A2 (cat. no. 21295‐1‐AP), Nanog (cat. no. 14295‐1‐AP), aldehyde dehydrogenase 1 (ALDH1) A1 (cat. no. 15910‐1‐AP), and β‐actin (cat. no. 60008‐1‐Ig) were purchased from Proteintech (Wuhan, Hubei, P.R.C). Primary antibodies against Gsk3β (ab93926), Wnt3a (ab28472) and β‐catenin (ab32572) were purchased from Abcam (Cambridge, MA, USA). The secondary antibodies (cat. no. KGAA35 and cat. no. KGAA37) were horseradish peroxidase‐conjugated and purchased from KeyGEN BioTECH. New Super ECL (cat. no. KGP1127; KeyGEN BioTECH) was used to develop image in Tanon 5200 machine (Tanon, Shanghai, China).
Nitrocellulose membrane
Manufactured by Promega
Sourced in United States
Nitrocellulose membranes are porous sheets made of chemically treated cellulose. They are used for the transfer and immobilization of proteins, nucleic acids, or other macromolecules in various analytical techniques, such as Western blotting, dot blotting, and slot blotting.
Automatically generated - may contain errors
Lab products found in correlation
Lysis buffer, by Keygen Biotech (2 mentions)
Bca protein assay kit, by Keygen Biotech (2 mentions)
Odyssey fc imaging system, by LI COR (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ab93926, by Abcam (1 mentions)
Ab28472, by Abcam (1 mentions)
Ab32572, by Abcam (1 mentions)
Ab185966, by Abcam (1 mentions)
Nano glo luciferase assay substrate, by Promega (1 mentions)
Protease inhibitor mixture, by Merck Group (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Ab179484, by Abcam (1 mentions)
Ma5 15738, by Thermo Fisher Scientific (1 mentions)
Ripa lysis buffer, by Merck Group (1 mentions)
Proliferating cell nuclear antigen pcna, by Cell Signaling Technology (1 mentions)
Nupage bistris gels 4 12, by Thermo Fisher Scientific (1 mentions)
Glyceraldehyde 3 phosphate dehydrogenase gapdh, by Merck Group (1 mentions)
Irdye 680, by LI COR (1 mentions)
Irdye 800, by LI COR (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
5 protocols using nitrocellulose membrane
1
Western Blot Analysis of Stem Cell Markers
2
Western Blot Analysis of Stem Cell Markers
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The detailed procedure was mentioned in a previous study 20 . Briefly, cells were lysed using lysis buffer (KeyGEN BioTECH, Nanjing, China). Protein concentration was determined using the BCA Protein Assay Kit (KeyGEN BioTECH). 20 μg of protein was separated by 10% SDS/PAGE, then transferred to nitrocellulose membrane (Promega, Madison, WI, USA) and incubated with the primary antibody against SOX9 (ab185966), which was purchased from Abcam, primary antibodies against Nanog (cat. no. 14295‐1‐AP), aldehyde dehydrogenase 1 (ALDH1) A1 (cat. no. 15910‐1‐AP), E‐cadherin (cat. no. 20874‐1‐AP), vimentin (cat. no. 10366‐1‐AP) and β‐actin (cat. no. 66009‐1‐Ig) were purchased from Proteintech (Wuhan, Hubei, China). The secondary antibodies (cat. no. KGAA35 and cat. no. KGAA37), horseradish peroxidase conjugated, were purchased from KeyGEN BioTECH. Images were developed using New Super ECL (cat. no. KGP1127; KeyGEN BioTECH). Protein expression levels were quantified by density analysis using quantity one (New Brunswick, NJ, USA) Software and normalized to levels of β‐actin.
3
Protein Isolation and Western Blot Analysis
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Cells were lysed using radioimmunoprecipitation assay (RIPA) lysis buffer (15 mM NaCl, 1 mM MgCl2, 1 mM MnCl2, 2 mM phenylmethylsulfonyl fluoride and protease inhibitor mixture [MilliporeSigma, USA]), lysates mixed 5:1 with 6× sample solubilizer (0.0625 M Tris·HCl (pH 6.8), 10% glycerol, 0.01% bromophenol blue, 2% (w/v) SDS, 2% 2-mercaptoethanol), and then heated at 95 °C for 5 min. Proteins were electrophoresed on discontinuous Laemmli polyacrylamide gels, transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA), and probed with primary antibodies and secondary HRP-conjugated antibodies and antibodies visualized by chemiluminescence (Thermo Fisher Scientific) as per manufacturer’s instructions. HiBiT-tagged proteins were identified by probing nitrocellulose membranes for 1 min with LgBiT protein in the presence of Nano-Glo luciferase assay substrate (Promega), followed by chemiluminescent visualization. Image processing was performed using FlourChem E (Protein Simple).
4
Protein Extraction and Western Blot Analysis
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Total proteins were extracted from cells with RIPA lysis buffer (Sigma-Aldrich) supplemented with protease inhibitor cocktail (Sigma-Aldrich), and 20–30 μg of proteins were size-separated using NuPage BisTris gels 4–12% (Invitrogen) and transferred to nitrocellulose membranes (Promega). Membranes were blocked with PBS containing 0.1% Tween-20 and 5% non-fat dry milk and incubated with antibodies to CCDC141 (1:1000 dilution, SAB3500670, Sigma-Aldrich), Ac-tubulin (1:1000 dilution, ab179484, Abcam), Proliferating Cell Nuclear Antigen (PCNA 1:2000 dilution, 2586, Cell Signaling Technologies) and Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH 1:10,000 dilution, G9545, Sigma-Aldrich and MA5-15738, Thermofisher). Anti-rabbit and mouse IRDye680 or IRDye800 (Licor) were used as secondary antibodies (1:10,000 dilution). Immunoblots were scanned with the Odyssey® Fc Imaging System (Licor).
5
Western Blot Analysis of EAP1 Protein
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Forty-eight hours post-transfection cells were harvested and lysed in radio-immunoprecipitation assay buffer (Sigma Aldrich) supplemented with protease inhibitor (Roche Diagnostics Ltd, West Sussex, UK). The concentration of the cell lysates was measured by the Pierce BCA protein assay kit (Thermo Fischer Scientific) according to the manufacturer’s instructions. Equal amount of proteins was separated by standard deviation score (SDS)-PAGE (4–12% polyacrylamide NuPage Bis–Tris gels; Invitrogen) and transferred on nitrocellulose membranes (Promega, Southampton, UK). Non-specific binding was blocked with 5% non-fat milk in phosphate buffered saline (PBS) containing 0.1% Tween-20 (PBT). Membranes were incubated overnight at 4°C with primary antibodies diluted in 5% non-fat milk in PBT: rabbit polyclonal anti-EAP1 diluted at 1:2000 (Sigma Genosys) and mouse monoclonal anti-GAPDH diluted at 1:5000 (Santa Cruz Biotechnology, Heidelberg, Germany). After washes in PBT, the membranes were probed with secondary antibodies (1:10 000; Licor, Cambridge, UK). After washes in PBT, the membranes were scanned and analyzed using the Odyssey Fc Imaging System (Licor). Experiment was repeated three independent times.
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