Smad4 Antibody (B-8): sc-7966

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	-Mouse (1:1000) -Use clean forceps to gently handle the blot from the corner without creasing the membrane. Do not write on the blot with pen or marker, as the ink can fluoresce and cause background.

Protocol tips
-Mouse (1:1000) -Use clean forceps to gently handle the blot from the corner without creasing the membrane. Do not write on the blot with pen or marker, as the ink can fluoresce and cause background.

Publication protocol

Quantitative immunoblotting was performed as previously described (Janes, 2015). Samples were prepared in dithiothreitol-containing Laemmli sample buffer to a total volume of 20 or 40 μl. Polyacrylamide gels (8, 10, 12, or 15%) of 1.5-mm thickness were cast, and samples were electrophoresed in tris-glycine running buffer (25 mM tris base, 250 mM glycine, and 0.1% SDS) at 130 V. Proteins were transferred to a PVDF membrane (Millipore; Immobilon-FL, 0.45 μm pore size) in a Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad) in transfer buffer (25 mM tris, 192 mM glycine, 0.0375% SDS, and 10–40% methanol) at 100 V for 1 hr on ice. Membranes were blocked with 0.5× Odyssey blocking buffer in PBS (fluorescence detection) or 5% nonfat skim milk in TBS-T (chemiluminescence detection). Primary antibodies were diluted with 0.5× Odyssey blocking buffer + 0.1% Tween-20 (fluorescence detection) or with 5% BSA in TBS-T (chemiluminescence detection).Membranes were washed and probed with secondary antibody diluted with 0.5× Odyssey blocking buffer (fluorescence detection) or with 5% nonfat skim milk in TBS-T (chemiluminescence detection). Membranes for fluorescence imaging were scanned on an Odyssey infrared scanner (LI-COR) at 169-μm resolution and 0-mm focus offset. Membranes for chemiluminescence were covered with SuperSignal West Femto Reagent (Thermo Fisher #34096) and chemiluminescent exposures were captured on a ChemiDoc MP gel imager (Bio-Rad) with “Chemi Hi Resolution” settings. Raw 16-bit images of exposures were analyzed in ImageJ with the gel analysis tool. Four independent samples provide >80% power to detect a 50% change in mean assuming a coefficient of variation of 20% and noncentral t statistics with a type I error rate of α = 0.05.

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