Total protein extracts were prepared from cells using cell-lysis buffer containing a protease-inhibitor cocktail (aprotinin, leupeptin, pepstatin A, and phenylmethylsulfonyl fluoride). The protein concentration was determined by a Bradford protein-assay kit (Bio-Rad Laboratories Inc., Hercules, CA, USA). Equal amounts of total protein (40 μg) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (10%), followed by electrophoretic transfer to nitrocellulose membranes (GE Healthcare UK Ltd., Little Chalfont, UK). The blots were incubated with primary antibody (rabbit antirat) at 4°C overnight, followed by secondary antibody (horseradish peroxidase-conjugate goat antirabbit IgG) for 2 hours at room temperature. The bound antibodies were detected by the use of a SuperSignal Western blotting kit (Thermo Fisher Scientific). Densitometric analysis of protein bands was performed with Quantity One® software (version 4.5.2; Bio-Rad Laboratories Inc.,).
Supersignal western blotting kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The SuperSignal Western blotting kit is a laboratory product designed to facilitate the detection and visualization of proteins during Western blotting analysis. It provides the necessary reagents and components to enable the chemiluminescent detection of target proteins on a membrane.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by GE Healthcare (2 mentions)
Bradford protein assay kit, by Bio-Rad (2 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Ab6302, by Abcam (1 mentions)
Ab185736, by Abcam (1 mentions)
Gapdh antibody, by Beyotime (1 mentions)
Ring1b, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
Sds page gel, by Bio-Rad (1 mentions)
4 protocols using supersignal western blotting kit
1
Western Blot Protein Analysis Protocol
2
Western Blot Analysis of ZNRF3, β-Catenin, and TCF-4
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Total protein was extracted from tissue samples or cultured cells using RIPA lysis buffer supplemented with protease and phosphatase inhibitors. Protein concentration was determined using a Bradford Protein Assay kit (Bio-Rad Laboratories Inc., Hercules, CA, USA). Equal amounts of total protein (30 μg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (10%), followed by electrophoretic transfer onto nitrocellulose membranes (GE Healthcare UK Ltd, Little Chalfont, UK). The blots were incubated overnight at 4°C with primary antibody, rabbit antihuman polyclonal to ZNRF3-C-terminal, anti-β-catenin rabbit polyclonal antibody (ab6302), and rabbit polyclonal TCF-4 antibody (ab185736) (the proportion of dilution was 1:800, 1:600, and 1:800, respectively; Abcam, Cambridge, UK), followed by polymeric HRP-labeled antirabbit immunoglobulin G (the secondary antibody; Promega Corporation, Fitchburg, WI, USA) for 2 hours at room temperature. The bound antibodies were detected using the SuperSignal Western Blotting Kit (Thermo Fisher Scientific). To provide a quality control, GAPDH antibody (Beyotime Institute of Biotechnology, Haimen, Jiangsu, People’s Republic of China) was used as the internal control in each group.
3
Cell Lysis and Protein Extraction Protocol
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Whole cell lysates prepared in RIPA (radioimmunoprecipitation) assay buffer (1 × PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 0.1 mg/ml phenylmethanesulfonyl fluoride, 20 mM sodium fluoride, 0.2 mM sodium orthovanadate, and Complete Protease Inhibitor Mix, one tablet per 50 ml) [20 (link)-22 (link)]. Cytoplasmic and nuclear fractions were prepared as described previously [20 (link)-22 (link)]. Protein samples were separated on SDS-PAGE gel and transferred to nitrocellulose membranes, which were then incubated with the primary antibodies. After incubation with appropriate secondary antibodies, the immunoblots were developed using SuperSignal Western blotting kits (Pierce Biotechnology) and exposed to X-ray film according to the manufacturer’s protocol. Western blots were stripped between hybridizations with stripping buffer [10 mM Tris–HCl (pH 2.3) and 150 mM NaCl].
4
Western Blot Analysis of Chromatin Regulators
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