CRC tissue samples and cells were washed with ice-cold phosphate-buffered saline (PBS) 3 times and lysed in RIPA buffer supplemented with protease inhibitor. The protein concentration was determined using the bicinchoninic acid method. Equal amounts of proteins (30 µg) were separated by 15% SDS-PAGE and transferred to a PVDF membrane (Immobilon; Millipore Corp., Bedford, MA, USA). After blocking with 5% non-fat milk (diluted with phosphate-buffered saline/Tween-20, TBST) at 37°C for 1 h, the membranes were incubated with primary antibodies: anti-LRH-1 (1:1000, ab41901), anti-matrix metalloproteinase (MMP)2 (1:1,000, ab80737), anti-MMP9 (1:1,000, ab119906), anti-β-catenin (1:500, ab92514), cyclin D1 (1:1,000, ab134175), c-Myc (1:500, ab51156) and GAPDH (1:1,000, ab8245; Abcam, Cambridge, UK) at 4°C overnight. After rinsing with TBST, they were incubated with horseradish peroxidase-conjugated goat anti-mouse or rabbit immunoglobulin G antibodies (1:200; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Protein bands were detected using an enhanced chemiluminescence reagent (Pierce Biotechnology, Inc., Rockford, IL, USA). Band intensity was quantified using Image software (Media Cybernetics, Inc., Rockville, MD, USA).
Ab92514
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab92514 is a lab equipment product offered by Abcam. It is a device designed for use in research and scientific applications. The core function of this product is to perform a specific task required in laboratory settings. However, a detailed description of its intended use or features is not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
Ab8245, by Abcam (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ab80737, by Abcam (1 mentions)
Ab41901, by Abcam (1 mentions)
Ab51156, by Abcam (1 mentions)
Ab119906, by Abcam (1 mentions)
Ab134175, by Abcam (1 mentions)
Immobilon, by Merck Group (1 mentions)
Image software, by Media Cybernetics (1 mentions)
Enhanced chemiluminescent detection kit, by Thermo Fisher Scientific (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Ab76011, by Abcam (1 mentions)
Ab18956, by Abcam (1 mentions)
Ab22035, by Abcam (1 mentions)
Ab27798, by Abcam (1 mentions)
Ab24925, by Abcam (1 mentions)
Ab138378, by Abcam (1 mentions)
Ab119752, by Abcam (1 mentions)
Ab6721, by Abcam (1 mentions)
Quantity one software version 4, by Bio-Rad (1 mentions)
5 protocols using ab92514
1
Western Blot Analysis of Colorectal Cancer Markers
2
CTNND1 Protein Detection by Western Blot
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RIPA lysis buffer (Beyotime, Shanghai, China) was used to extract protein. Then, 50 μg was separated by 10% sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE), transferred to polyvinylidene fluoride (PVDF) membranes which were probed overnight at 4°C with primary antibodies: CTNND1 (1:500, Abcam, ab92514) and GAPDH (1:3000, Abcam, ab8245). Membranes were then probed for 2 h at room temperature with a horse radish peroxidase conjugated secondary antibody. Protein signals were detected using an enhanced chemiluminescent detection kit (Pierce Biotechnology, Rockford, IL, USA).
3
Comprehensive Antibody Analysis Protocol
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Antibodies used are as follow; N-cadherin antibody (ab76011, Abcam, Cambridge, UK), proN-cadherin antibody (GTX101141, GeneTex, San Antonio, TX, USA), GDNF antibody (ab18956, Abcam, Cambridge, MA, USA), N-cadherin (phospho Y860) (ab119752, Abcam), p120-catenin (ab92514, Abcam), β-catenin (GTX22982, Genetex, San Antonio, TX, USA), β-catenin phosphorylated antibodies including phospho Y654 (ab24925, Abcam), phospho Y489 (ab138378, Abcam), phospho Y142 (ab27798, Abcam), Ser33/37/Thr41 (9561, CST, Danvers, MA, USA), nestin (ab22035, Abcam, Cambridge, MA, USA), rabbit anti-CD133 (ab19898, proteintech, America), and anti-Caveolin-1 (SAB4200216, Sigma, Aldrich, USA).
4
Western Blot Analysis of CTNND1 Protein
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At 48 hours after transfection, cells were lysed using ice-cold RIPA buffer (Beyotime Biotechnology, Shanghai, People’s Republic of China) containing protease inhibitor cocktail (Hoffman-La Roche Ltd, Basle, Switzerland) to extract proteins. Protein concentrations were determined using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Then, proteins were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene fluoride (PVDF) membranes. After blocking with 5% non-fat milk, the membranes were probed overnight at 4°C with anti-CTNND1 antibody (ab92514, 1:1,000, Abcam, Cambridge, UK) or anti-β-actin antibody (ab8227, 1:2,000, Abcam). Next, the blots were subsequently incubated for 1 hour at room temperature with Goat Anti-Rabbit IgG H&L (HRP) secondary antibody (ab6721, 1:2,000, Abcam). At last, chemoluminescent signals were visualized using Super Signal™ West Dura Extended Duration Substrate (Thermo Fisher Scientific) and quantified via Quantity One Software Version 4.1.1 (Bio-Rad Laboratories, Hercules, CA, USA).
5
Immunohistochemical Analysis of Mouse Stomach
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After the mice had been killed, mouse stomachs were resected and fixed in 10 % neutral buffered formalin, and were then dipped into series of ethanol solutions (70–100 %) and embedded in paraffin. Immunohistochemistry (IHC) was conducted as described previously [19 (link)] using the following antibodies: anti-mouse β-catenin (1:1500, C2206, rabbit antiserum, Sigma-Aldrich, Tokyo, Japan), anti-mouse Ki-67 (2 μg/ml, ab15580, rabbit polyclonal antibody, Abcam, Tokyo, Japan), anti-mouse STAT3 (1:500, CST Stat3; 124H6, mouse monoclonal antibody; Cell Signaling Technology Japan, Tokyo, Japan), anti-mouse pSTAT3 (1:100, Tyr705; D3A7, XP rabbit monoclonal antibody, Cell Signaling Technology Japan), anti-mouse p53 protein (1:2,000, CM5, rabbit polyclonal antibody, Vector Laboratories, Burlingame, CA, USA), anti-mouse c-Myc (1:200, 9E10; sc-40, mouse monoclonal antibody, Santa Cruz Biotechnology, Dallas, TX, USA), and anti-mouse δ1-catenin [1:200, EPR357(2), ab92514, rabbit monoclonal antibody, Abcam]. IHC for β-catenin and Ki-67 was conducted by Genostaff (Tokyo, Japan). IHC for CD44 and F4/80 (MCA497R clone A3-1, Serotec, Oxford, UK) was conducted as described previously [11 (link), 26 (link)].
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