cyclin E Antibody (E-4): sc-377100

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

Following treatment with BHP, the T24 cells were washed with PBS and lysed in ice-cold lysis buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM ethylene glycol tetraacetic acid, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM sodium orthovandate and 1 mM NaF. The cell lysates were centrifuged at 12,000 × g for 45 min at 4°C to collect the supernatant. A Bradford protein assay was used to determine the protein concentration. The proteins (40 µg) were separated using 12% SDS-PAGE and then transferred onto a polyvinylidene difluoride membrane (EMD Millipore, Billerica, MA, USA). The membranes were blocked using 5% non-fat milk, followed by overnight incubation with primary antibodies at 4°C. The primary antibodies used were as follows: Cyclin E (1:1,000; cat. no. sc-377100), cell division cycle 25C (Cdc25c; 1:1,000; cat. no. sc-13138), p21 (1:1,00; cat. no. sc-6246), Bcl-2-associated X protein (Bax; 1:1,000; cat. no. sc-526), apoptosis-inducing factor (AIF; 1:1,000; cat. no. sc-5586), caspase-3 (1:1,000; cat. no. sc-7148), and poly(ADP-ribose) polymerase (PARP; 1:2,000; cat. no. sc-7150) all obtained from Santa Cruz Biotechnology, Inc., and phosphorylated (p)-p53 (1:50; cat. no. PA5-27822), B cell lymphoma-2 (Bcl-2; 1:200; cat. no. PA1-28275), caspase-9 (1:1,000; cat. no. KHZ0102) and caspase-8 (1:1,000; cat. no. MA1-41280) all obtained from Thermo Fisher Scientific, Inc., and endonuclease G (Endo G; 1:1,000; Koma Biotech, Seoul, Korea). Following incubation with primary antibodies, the membranes were washed with Tween and PBS and then incubated again for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibody (1:3,000; Bio-Rad Laboratories, Inc., Hercules, CA, USA; cat. no. 170–6515). Electrochemiluminesence reagent (GE Healthcare Life Sciences, Amersham, UK) was used to analyze the bound antibody complexes and β-actin (1:1,000; Santa Cruz Biotechnology, Inc.; cat. no. sc-47778) served as an internal control. A gel imaging analysis system (Kodak ID; Kodak, Rochester, NY, USA) was used to analyze the bands.

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Manufacturer protocol

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