Beclin-1 (D40C5) Rabbit mAb

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	1:1000 dilution for primary antibody

Protocol tips
1:1000 dilution for primary antibody

Publication protocol

The treated hFOB 1.19 cells were collected and lysed. The extract was centrifuged (20,000 × g, at 4°C for 20 min), and the supernatant was collected to harvest total protein. The protein concentration in the extract was determined using the BCA protein assay (Wuhan Boster Biological Technology, Ltd., Wuhan, China). An equal amount of protein was resolved using SDS-PAGE and electroblotted onto a PVDF membrane (Millipore, Billerica, MA, USA). The membrane was then incubated overnight at 4°C with primary antibody against LC3 (1:1,000), beclin 1 (1:1,000) and GAPDH (1:2,000), respectively. Next, the membrane was incubated with goat anti-rabbit secondary antibody conjugated to horseradish peroxidase (1:5,000; ZSGB-BIO, Beijing, China) for 1 h at room temperature. The protein signal was enhanced using a chemiluminescene system (DNR MF-ChemiBIS 3.2). The band density was quantified using the ImageJ image processing program.

Immunofluorescence staining
For immunofluorescence detection, 8×103 hFOB 1.19 cells were seeded in a 24-well plate and treated with indicated drug for 6 h. The hFOB 1.19 cells were then incubated with anti-LC3 antibody (1:200) at 4°C overnight. The secondary antibody, Alexa Fluor® 488 Goat Anti-Rabbit IgG (ZSGB-BIO), was applied at room temperature for 1 h, followed by staining the nuclei with DAPI (Beyotime Institute of Biotechnology). Cells were photographed using the inverted fluorescence microscope. LC3-positive cells, defined as cells with visible LC3 foci, were quantified by manually in three randomly-selected fields; nuclei were enumerated by counting DAPI-stained nuclei in the same field. The number of LC3-positive cells in each microscopic field was divided by the number of nuclei in the same field, and was regarded as the rate of LC3-positive cells.

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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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