Santa Cruz Biotechnology
Anti-Rad17 (H-300) antibody
sc-5613
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Cells were washed twice in PBS and harvested with lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerol phosphate, 1 mM Na3VO4, 1µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride) on ice for 10 min. The supernatants were collected after centrifugation at 12,000 g for 30 min. The protein concentrations were determined using the BCA protein assay kit (Pierce, Rockford, IL). Equal amounts of protein lysates were separated by SDS-PAGE and transferred onto nitrocellulose membranes, followed by incubation with primary antibody at 4°C overnight. After washing, the membranes were incubated with HRP-conjugated secondary antibodies at room temperature for 1 hour. Proteins were detected with the enhanced chemiluminescence (ECL) kit (Pierce, Rockford, IL).
Western Blot
Various types of breast cancer cell lines
4 degrees Celsius overnight
5% milk
Goat anti-rabbit secondary antibody
Room temperature 1 hour
ECL
Elevated Rad17 expression in various types of breast cancer cell lines in comparison with breast epithelial cell lines, indicating unexpected accumulation of Rad17 in breast cancer cells comparing with breast epithelial cells
See Figure 1A at http://www.jbc.org/content/288/25/18134.full.pdf+html?with-ds=yes
Works very well
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Works beyond expectation