BECN1 Antibody (H-300)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- Lyse cells in RIPA lysis buffer containing a complete protease inhibitor mixture and a protein phosphatase inhibitor.	- Dilute Primary Ab -1:1000 - Incubate antibody overnight at 4°C

Upstream tips
- Lyse cells in RIPA lysis buffer containing a complete protease inhibitor mixture and a protein phosphatase inhibitor.
Protocol tips
- Dilute Primary Ab -1:1000 - Incubate antibody overnight at 4°C

Publication protocol

The expression and phosphorylation level of target proteins were detected by western-blot analysis. Cells were lysed with RIPA lysis buffer containing a complete protease inhibitor mixture and a protein phosphatase inhibitor. After protein extraction, the cell lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to PVDF membranes. Nonspecific binding was blocked using 5% BSA, and the membranes were then incubated with specific primary antibodies overnight at 4°C. After incubating the membrane with the appropriate secondary antibodies conjugated to horseradish peroxidase, Super Enhanced chemiluminescence detection reagents were used to detect the specific bands. The immunoblots were quantified by densitometric analysis using Quantity One software (Bio-Rad, Hercules, USA). The following primary antibodies (1:1000) were used: anti-GRP78, anti-CHOP, anti-Beclin-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-LC3B (Cell Signaling Technology, Danvers, MA, USA). GAPDH was used as an internal control.

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Manufacturer protocol

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