Beclin-1 (D40C5) Rabbit mAb

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Dilute primary Ab at 1:1,000 and incubate overnight at 4C.

Protocol tips
Dilute primary Ab at 1:1,000 and incubate overnight at 4C.

Publication protocol

Western blot analysis was performed according to our previous protocol [41]. Briefly, both vascular tissue samples obtained from ApoE-/- mice and THP-1-derived macrophages treated with PBS or ox-LDL were harvested, and added to Cell Lysis Buffer for Western and IP with both protease and phosphatase inhibitors following the manufacturers’ instructions. The homogenate was centrifuged at 14,000 x g for 15 min at 4°C. The protein concentration was measured using a BCA protein assay kit according to the manufacturer's protocol. Same amounts of total protein were loaded in each lane of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) using 12% gels and then were transferred onto a polyvinylidene difluoride (PVDF) membrane. The membranes were blocked for 4 hrs with 5 % (w/v) non-fat milk powder in Tris-buffered saline containing 0.1% (v/v) Tween-20 (TBST) at room temperature and incubated overnight with the primary antibodies at 4°C. The membranes were washed with TBST, incubated with appropriate HRP-conjugated secondary anti-mouse or -rabbit antibodies for 2 hrs at room temperature, and rewashed with TBST for 30 min (three times for 10 min each). An enhanced chemiluminescence solution was used to develop the blots for 5 min. Target proteins were detected using an enhanced chemiluminescence detection system (MiniChemi, Beijing Sage Creation Science Co., Ltd., Beijing, China). Pre-determined molecular weight standards were used as markers. Relative protein expression levels were semiquantitatively performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

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Manufacturer protocol

Download the product protocol from Cell Signaling Technology for Beclin-1 (D40C5) Rabbit mAb below.

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