Expression levels of EGFR, VDR and phosphorylated forms of EGFR and Akt in tissues were measured by immunoblotting of whole tongue extracts using primary antibodies specific for these markers. Western blots were performed using the following primary antibodies: polyclonal rabbit anti-EGFR (1005; Santa Cruz, Dallas, TX), monoclonal rat anti-VDR (clone: 9A7; Thermo Fisher Scientific, Waltham, MA), monoclonal rabbit anti-phospho-EGFR (phospho Y1092; abcam, Cambridge, MA), polyclonal rabbit anti-phospho-AKT1 (phospho S473; abcam, Cambridge, MA). Anti-rabbit and anti-mouse horseradish-peroxidase-conjugated secondary antibodies (GE Healthcare, Pittsburgh, PA) were used and blots were developed using enhanced chemiluminescence. Actin (I-19; Santa Cruz, Dallas, TX) was used in each experiment as a loading control. To assess difference in protein expression, western blot films were analyzed using the UN-SCAN-IT program (Silk Scientific, Inc., Orem, UT). Equal sized regions of interest (ROIs) were fitted around each expression band to measure total pixels. Using the total pixels acquired from the blot and the total pixels acquired from the corresponding actin blot, a normalized expression value was achieved [(antibody total pixels/actin total pixels)/average control tissue expression].
Anti rabbit and anti mouse horseradish peroxidase conjugated secondary antibodies
Manufactured by GE Healthcare
Anti-rabbit and anti-mouse horseradish-peroxidase-conjugated secondary antibodies are laboratory reagents used in immunodetection techniques. They bind to primary antibodies raised against rabbit or mouse antigens and facilitate their detection through the enzymatic activity of horseradish peroxidase.
Automatically generated - may contain errors
Lab products found in correlation
Actin 1 19, by Santa Cruz Biotechnology (1 mentions)
Polyclonal rabbit anti egfr 1005, by Santa Cruz Biotechnology (1 mentions)
Ecl western blotting detection system, by GE Healthcare (1 mentions)
Amersham hybond p, by GE Healthcare (1 mentions)
Image studio software, by LI COR (1 mentions)
Sodium orthovanadate, by Merck Group (1 mentions)
2 protocols using anti rabbit and anti mouse horseradish peroxidase conjugated secondary antibodies
1
Quantifying Protein Expression Levels in Tongue
2
Western Blot Analysis of CD133 in Cells
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Cells were lysed in RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 1 mM EDTA, 0.5% sodium deoxycholate, 0.1% SDS) with protease inhibitors, 1 mM PMSF, 1 mM DTT, and 0.5 mM sodium orthovanadate (Sigma-Aldrich). Protein concentration was determined by the Bradford assay and 40 mg of proteins per sample were resolved on an 8% SDS-PAGE gel and blotted onto a PVDF membrane (Amersham HyBond-P GE Healthcare, UK). After blocking at RT in 5% dry-milk in TBS containing 0.1% Tween-20 for 1 h, membranes were incubated overnight at 4°C with rabbit polyclonal anti-CD133 antibody (dilution 1:500, abcam 19898), and mouse monoclonal anti-b-actin (dilution 1:2000) antibody was used for normalization. Membranes were then incubated with anti-rabbit and anti-mouse horseradish peroxidase conjugated secondary antibodies (dilution 1:10000, GE Healthcare Bio-Sciences, Piscataway, NJ). Immunocomplexes were detected by ECL Western Blotting detection system (GE Healthcare Bio-Sciences). Densitometric analysis was performed with Image Studio software (LI-COR Biosciences, Lincoln, NE).
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