Beclin-1 Antibody

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- Lyse cells with RIPA lysis buffer containing Tris-HCl 10 mmol/L, EDTA 5 mmol/L, NaCl 50 mmol/L, 1% deoxycholic acid and 1% triton X-100, pH 7.4	- Dilute Primary Ab -1:2000.

Upstream tips
- Lyse cells with RIPA lysis buffer containing Tris-HCl 10 mmol/L, EDTA 5 mmol/L, NaCl 50 mmol/L, 1% deoxycholic acid and 1% triton X-100, pH 7.4
Protocol tips
- Dilute Primary Ab -1:2000.

Publication protocol

Cell cultures were lysed with RIPA lysis buffer (Tris-HCl 10 mmol/L, EDTA 5 mmol/L, NaCl 50 mmol/L, 1% deoxycholic acid and 1% triton X-100, pH 7.4). Protein concentration was determined by the Bradford method (BioRad protein assay). Equal amounts of proteins from cell extracts were separated by SDS-PAGE 8–15%, electro-transferred to PVDF membranes and blocked with 5% milk. Primary antibodies were used against type I collagen 1:2000 (cat #ABT123 Millipore), p62 1:2000 (cat #5114 Cell Signaling), osteopontin 1:2000 (cat #ab8448 Abcam), Beclin1 1:2000 (cat #3738 Cell Signaling), α-SMA 1:20000 (cat #ab7817 Abcam), GAPDH 1:50000 (cat #8795 Sigma), SM22 1:10000 (cat #ab14106 Abcam), LC3-I/II 1:1000 (cat #2775 Cell Signaling). Membranes were re-blotted with a horseradish peroxidase-linked secondary antibody 1:5000 (mouse cat #402335 and rabbit cat#401315 Merck). Bands were detected using ECL (Biological Industries) and luminescence was assessed using a digital imaging system (Syngene). Quantification of the bands by densitometry was performed using UN-SCAN-IT gel software.

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Manufacturer protocol

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