Sigma-Aldrich
Anti-β-actin antibody
A2228
Browse Similar Products: Anti-beta-Actin Antibodies
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).
Research Published: Zhou Z, Chao J, Zhang L, Takeo F, Kim H, Huang Y, Liu Z, Wan Y. 2013. Regulation of Rad17 turnover unveils an impact of Rad17-APC cascade in breast carcinogenesis and treatment. J Biol Chem, 288(25):18134-45.
Western Blot
Various breast cancer cell lines
4 degrees celcius overnight
5% milk
Goat anti-mouse secondary antibody
Room temperature 1 hour
ECL
Clear specific bands
See Figure 1A at http://www.jbc.org/content/288/25/18134.full.pdf+html?with-ds=yes
Works very well
NA
Works beyond expectation