Beclin-1 (D40C5) Rabbit mAb

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Lyse cells in pre-cooled RIPA lysis buffer containing 50 mM Tris-HCl (pH 7.4) 150 mM NaCl, 1 mM dithiothreitol (DTT), 0.25% sodium deoxycholate, 0.1% NP-40, 1 mM phenylmethysulfonyl fluoride (PMSF), 50 mM sodium pyrophosphate, 1 mM Na3VO4, 1 mM NaF, 5 mM EDTA, 5 mM EGTA and a protease inhibitor cocktail.	Dilute primary Ab at 1:1000 and incubate overnight at 4ºC

Upstream tips
Lyse cells in pre-cooled RIPA lysis buffer containing 50 mM Tris-HCl (pH 7.4) 150 mM NaCl, 1 mM dithiothreitol (DTT), 0.25% sodium deoxycholate, 0.1% NP-40, 1 mM phenylmethysulfonyl fluoride (PMSF), 50 mM sodium pyrophosphate, 1 mM Na3VO4, 1 mM NaF, 5 mM EDTA, 5 mM EGTA and a protease inhibitor cocktail.
Protocol tips
Dilute primary Ab at 1:1000 and incubate overnight at 4ºC

Publication protocol

For the extraction of the total cell proteins, the cells were washed with pre-cooled PBS and subsequently lysed in a pre-cooled RIPA lysis buffer containing 50 mM Tris-HCl (pH 7.4) 150 mM NaCl, 1 mM dithiothreitol (DTT), 0.25% sodium deoxycholate, 0.1% NP-40, 1 mM phenylmethysulfonyl fluoride (PMSF), 50 mM sodium pyrophosphate, 1 mM Na3VO4, 1 mM NaF, 5 mM EDTA, 5 mM EGTA and a protease inhibitor cocktail. Cell lysis was performed on ice for 30 min. Clear protein extracts were obtained by centrifugation at 12,000 × g for 30 min at 4ºC. The protein extraction procedure from the MG63 cells was performed as previously described. The protein concentration was determined using a Bradford protein assay kit (KeyGen). The proteins were resolved on 8.5% polyacrylamide gels and subsequently transferred onto nitrocellulose membranes. For immunoblotting, the nitrocellulose membranes were incubated overnight at 4ºC with specific antibodies recognizing the target proteins. The membranes were then incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (1:3,000) for 1 h at room temperature and subsequently analyzed using an enhanced chemiluminescence (ECL) detection system (Amersham Pharmacia Biotech, Amersham, UK) and visualized by autoradiograpy. β-actin proteins (1:5,000; Sigma) were used as loading controls.

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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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