Cyclin E1 Antibody

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	-Rabbit (1:1000) -Soak PVDF membranewith pore size 0.45 um in methanol for 30 sec -Apply Wet transfersystems at 180mA for 90 min with Wet transfer buffer.

Protocol tips
-Rabbit (1:1000) -Soak PVDF membranewith pore size 0.45 um in methanol for 30 sec -Apply Wet transfersystems at 180mA for 90 min with Wet transfer buffer.

Publication protocol

HaCaT cells, treated with 1, 2 and 4 µM shikonin for 24 h, were lysed in radioimmunoprecipitation assay lysis buffer containing a protease and phosphatase inhibitor cocktail on ice for 30 min. Then lysate was then collected and centrifuged at 56,000 × g for 15 min at 4°C. Protein concentration was determined using the Bicinchoninic Acid Protein Quantification kit (Pierce Biotechnology, Inc., Rockford, IL, USA), according to the manufacturer's protocol. Protein lysates were then denatured for 10 min at 95°C and 50 µg protein per lane was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes (EMD Millipore, Billerica, MA, USA). After immunoblotting, the PVDF membranes were blocked with 5% skimmed milk for at least 2 h, washed and incubated with anti-caspase 3, anti-Akt, anti-p-Akt, anti-Erk 1/2, anti-p-Erk 1/2, anti-cyclin B1, anti-cyclin D1, anti-cyclin E, anti-Bcl-2, anti-Bax, anti-Bak, anti-GAPDH and anti-β-actin primary antibodies at 4°C overnight (all 1:1,000). Subsequently, the membranes were washed three times with Tris-buffered saline with Tween 20 and incubated with the HRP-conjugated goat anti-rabbit IgG (H+L) secondary antibodies (1:10,000) at 37°C for 1 h. Chemiluminescence detection was assayed using an enhanced chemiluminescence detection kit (Pierce Biotechnology, Inc.). Results were analyzed using Quantity One software (version 4.4.0.36; Bio-Rad Laboratories, Inc.), in order to obtain the optical density ratio of the target protein to GAPDH and β-actin.

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