This review of our SCO1 antibody comes from Fitzgerald customer Aren Boulet at the University of Saskatchewan. Aren writes:
Pieces of whole liver from liver-specific Sco1 knockout mice (SCO1liv/liv) and
wild-type littermates (SCO1wt/wt) were extracted in RIPA buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 1% Triton X-100, 0.5% deoxycholate and 0.1% sodium dodecyl sulfate) in the presence of a complete protease inhibitor cocktail.
Samples were denatured for 10 minutes at 37°C in Laemmli buffer containing 100 mM dithiothreitol, and 20 μg of total protein was separated by SDS-PAGE on a 4-20% Tris-HCl gel. Following semi-dry transfer, the membrane was blocked for a minimum of 4 hours at room temperature in 20 mM Tris (pH 7.4), 135 mM sodium chloride and 0.05% Tween-20 (TBST) supplemented with 5% milk, and incubated overnight at 4°C in blocking buffer containing SCO1 antibody at a 1:1000 dilution.
The membrane was then washed with blocking buffer for 60 minutes, incubated for 1 hour at room temperature in blocking buffer containing secondary antibody, and washed again with blocking buffer for 60 minutes.
Finally, the membrane was washed 3 times in TBST, and developed by enhanced chemiluminescence. An immunoreactive band at the expected molecular weight of SCO1 was detectable in the SCO1wt/wt extract but not in the SCO1liv/liv extract.
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