Western blot analysis of extracts of various cell lines, using Anti-HFE Antibody (A11436) at 1:1,000 dilution. The secondary antibody was Goat Anti-Rabbit IgG H&L Antibody (HRP) at 1:10,000 dilution. Lysates/proteins were present at 25µg per lane. The blocking buffer used was 3% non-fat dry milk in TBST.
Experimental Conditions: WB: 30 mg lysate from human and mouse cell lines prepared fresh in RIPA buffer [10 mM Tris-Cl (pH 8.0), 1 mM EDTA, 140 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS] with protease inhibitor cocktail (1:50, Thermo Scientific, A32963); 10% PAGE; PVDF membrane; tank transfer at 120-180 V for 60 min; blocked in 5% non-fat milk in TBS-T for 1 h; primary antibody overnight at 4C; washed and incubated with secondary antibody.
Dilution: Primary: 1:500
Results Summary: Successful detection of HFE via western blot in human and mouse cell lines (DT, CRH, MIAPACA, A8184, and PANC1) using Anti-HFE antibody (A11436).
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