The cells were detached using cell scrapers and subsequently incubated on ice for a duration of thirty minutes. Following this, the cell lysates were subjected to boiling at a temperature of 100℃ for a period of 5–10 min. The total protein content was then separated by electrophoresis using SDS-PAGE and subsequently transferred onto PVDF membranes. The blots were cut prior to hybridisation with antibodies during blotting. Afterwards, it was incubated overnight at 4℃ with primary antibodies against FDX1 (Cat. no. #ab108257; 1:1000; abcam), β-tubulin (Cat. no. #M20005, 1:5000; abmart), Vimentin (Cat. no. T55134, 1:1000; abmart), E-cadherin (Cat. no. TA0131, 1:1000; abmart), N-cadherin (Cat. no. T55015; 1:1000; abmart). The PVDF membrane was washed 3times, each time 10 min with TBST-Tween 20, and then incubated for 1–2 h with goat anti-rabbit lgG secondary antibody and goat anti-mouse lgG secondary antibody (Affinity HRP; 1:5000). Finally, washed the PVDF membrane and visualized with chemiluminescence (Cat. no. 34577; Thermo Scientific™).
E cadherin
Manufactured by Abmart
Sourced in United States
E-cadherin is a cell-cell adhesion protein that plays a critical role in maintaining the structural integrity of epithelial tissues. It functions by facilitating the formation of adherens junctions between adjacent cells, which are essential for the regulation of cell-cell communication and tissue homeostasis.
Automatically generated - may contain errors
Lab products found in correlation
Vimentin, by Abmart (1 mentions)
β tubulin, by Abcam (1 mentions)
Protease inhibitor, by Beyotime (1 mentions)
Supersignal west pico chemiluminescent substrate kit, by Thermo Fisher Scientific (1 mentions)
Uvp chemstudio plus imager, by Analytik Jena (1 mentions)
Bca method, by Beyotime (1 mentions)
Horseradish peroxidase labeled goat anti rabbit igg h l, by Beyotime (1 mentions)
Cryostat microtome, by Leica camera (1 mentions)
Alexa fluor 488 coupled goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Axioskop 2 mot fluorescence microscope, by Zeiss (1 mentions)
Dapi reagent, by Thermo Fisher Scientific (1 mentions)
Zen microscopy software, by Zeiss (1 mentions)
3 protocols using e cadherin
1
Western Blot Protein Detection
2
Quantification of E-Cadherin Expression in Intestinal Tissue
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To determine the protein expression level of E-cadherin, the intestinal ileum tissue was extracted and lysed in 200 μl of RIPA buffer containing protease inhibitors (Beyotime, Shanghai, China) on the ice for 5 mins. Both the infected group and the control group contained 4 mouse samples at each time point. The lysed tissue was then homogenized using a tissue crusher and centrifuged at 15000 rpm, 4˚C for 10 mins. The protein concentration was measured using the BCA method (Beyotime). Samples were resolved by SDS-PAGE using 50 μg of protein each and transferred onto nitrocellulose membranes. The membranes were blocked with 5% skimmed milk and then probed with E-cadherin (Abmart) diluted in 1:1000, followed by horseradish peroxidase labeled goat anti-rabbit IgG (H+L) diluted in 1:1000 (Beyotime). The membranes were analyzed using the Super Signal West Pico Chemiluminescent Substrate Kit (ThermoFisher) and were scanned on the UVP ChemStudio PLUS Imager (Analytikjena).
3
Immunofluorescent Staining of E-cadherin
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The 4 μM sections were obtained using a cryostat microtome (Leica, Wetzlar, Germany) and placed onto glass slides. The paraffin sections underwent two rounds of dewaxing in xylene, followed by gradient alcohol treatment (100%, 90%, 80%, 70%) for 3 mins each. The sections were then washed with PBS and repaired with acid antigen repair solution at 100˚C for 10 mins. The sections were treated with 10% goat serum at room temperature for 1 hour, then incubated with E-cadherin (Abmart, Berkeley Heights, NJ, USA) diluted in 1:100 for overnight at 4˚C. Subsequently, the slices were incubated with Alexa Fluor 488 coupled goat anti-mouse IgG (ThermoFisher) diluted in 1:1000 and Sporo-Glo diluted in 1:50 at room temperature for 1 hour. The slides were rinsed with PBS and then treated with the Slowfade Gold anti-fading agent containing DAPI reagent (ThermoFisher) to the cover glass. The stained slides were examined under a Zeiss Axioskop Mot 2 fluorescence microscope (Carl Zeiss, Oberkochen, Germany). The images were processed using ZEN microscopy software (Carl Zeiss, Oberkochen, Germany).
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