FLO-1 parental cells were lifted using 0.5 mM EDTA/PBS mixture (30 min), filtered through a 40 μm nylon mesh, pelleted by centrifugation (1400 rpm, 4 min) and resuspended in blocking buffer (2% BSA and 2% FBS in PBS) for 1 hr at 4°C. Cells were then incubated in labeling buffer (2% BSA in PBS) with either anti-IgG (Dako) or anti-E-cadherin (EP700Y, Abcam) antibodies for 1 hr at 4°C. After two washes, cells were stained with Alexa Fluor 700 conjugated goat anti-rabbit IgG (Invitrogen) for 1 hr at 4°C in the dark. Cells were then washed thrice before the addition of 2% FluoroGold™ (Sigma-Aldrich). Viable FLO-1 cells were FACS sorted for E-cadherin high and low expressing populations, and separately injected into NSG mice (500,000 cells/mice). Animals were sacrificed once their subcutaneous tumours reached 1500 mm3 and lungs and livers were harvested for quantification of metastatic burden by PCR.
Anti e cadherin ep700y
Manufactured by Abcam
Anti-E-cadherin (EP700Y) is a rabbit monoclonal antibody that recognizes the E-cadherin protein. E-cadherin is a transmembrane glycoprotein that plays a crucial role in cell-cell adhesion and maintenance of epithelial tissue integrity.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using anti e cadherin ep700y
1
E-cadherin Expression and Metastasis
2
Western Blot Analysis of E-Cadherin
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Transfected cells were lysed with Radioimmunoprecipitation Assay Buffer (RIPA buffer), supplemented with protease inhibitors (SigmaFast™ protease inhibitor -S8820) and phosphatase inhibitors (1 mM sodium orthovanadate and 10 mM sodium fluoride). Protein extracts were quantitated using a Bio-Rad Protein Assay according to the manufacturer's instructions (Bio-Rad). 50 g of total protein per sample were resolved in 12% polyacrylamide gel by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were probed with anti-E-Cadherin (EP700Y -Abcam) and anti-GAPDH (ab9485 -Abcam). Bands were visualized on ImageQuant (GE Healthcare) by chemiluminescence using Enhanced chemiluminescent ECL™ Prime Western Blotting System (GE Healthcare) following the manufacturer's instructions.
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