Monoclonal Anti-Caveolin-1 antibody produced in mouse

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	-Mouse (1:500)

Protocol tips
-Mouse (1:500)

Publication protocol

The testes of male ICR mice were solubilized in lysis buffer (7 mol/L urea, 2 mol/L thiourea, 4% (W/V) CHAPS and 2% (W/V) DTT), in the presence of 1% (W/V) protease inhibitor cocktail (Pierce Biotechnology, Rockford, IL,USA), and were then homogenized and sonicated. The mixture was placed on a shaker at 4°C for 1 h, and insoluble matter was removed by centrifugation at 40,000 g for 1 h at 4°C. The protein concentration of each sample was quantified by the Bradford protein assay using bovine serum albumin as a standard.

Samples containing 100 μg protein were electrophoresed on 4–20% polyacrylamide gradient gels, followed by transfer onto polyvinylidene difluoride membranes (GE Healthcare, San Francisco, CA, USA), blocked in triethanolamine buffered saline (TBS) containing 5% non-fat milk for 2 hours. Membranes were incubated overnight at 4°C with 1:1000 anti-flotillin-2 (rabbit polyclonal, ab135389, Abcam, Hongkong, China), 1:500 anti-flotillin-1 (rabbit monoclonal, ab133497, Abcam, Hongkong, China), 1:500 anti-caveolin-1 (mouse monoclonal, SAB4200216, Sigma Aldrich, USA) and 1:2000 anti-GAPDH (mouse monoclonal, KC-5g5, Shanghai Kangchen Biotechnology Company, Shanghai, China) in TBS containing 5% non-fat milk powder. Expression of GAPDH was used as a loading control. The following day, blots were washed and incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Beijing Zhongshan Biotechnology Company, Beijing, China) and the protein bands were detected using the enhanced chemiluminescence (ECL) Western Blot Detection Kit and AlphaImager (GE Healthcare, San Francisco, CA, USA).

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