Western blot analysis of extracts from UACC-62 cells, untreated (-), UV-treated (100 mJ/cm2, 15 min recovery; +), or starved with Earle's Balanced Salt Solution (EBSS, 15 min; +), using Phospho-Beclin-1 (Ser90) Antibody (upper) or Beclin-1 (D40C5) Rabbit mAb #3495 (lower).
Western blot analysis of extracts from PC-3 cells, untreated (-) or treated with carbonyl cyanide 3-chlorophenylhydrazone (CCCP; 30 μM, 2 hr; +), using Phospho-Beclin-1 (Ser90) Antibody (upper) or Beclin-1 (D40C5) Rabbit mAb #3495 (lower).
Western blot analysis of extracts from ACHN cells, untreated (-) or UV-treated (100 mJ/cm2, 1 hr recovery; +), using Phospho-Beclin-1 (Ser90) Antibody (upper) or Beclin-1 (D40C5) Rabbit mAb #3495 (lower).
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody datasheet or product webpage for recommended antibody dilution.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised October 2016
Protocol Id: 10
Phospho-Beclin-1 (Ser90) Antibody recognizes endogenous levels of Beclin-1 protein only when phosphorylated at Ser90. The antibody may not react with Beclin-1 when dually phosphorylated at Ser90 and Ser93. Background bands of unknown origin were detected at 100, 35, and 10 kDa in some cell lines.Species Reactivity:
Polyclonal antibodies are produced by immunizing animals with a synthetic phospho-peptide corresponding to residues surrounding Ser90 of human Beclin-1 protein. Antibodies are purified by protein A and peptide affinity chromatography.
Autophagy is a catabolic process for the autophagosomic-lysosomal degradation of proteins activated in response to nutrient deprivation and in neurodegenerative conditions (1). One of the proteins critical to this process is Beclin-1, the mammalian orthologue of the yeast autophagy protein Apg6/Vps30 (2). Beclin-1 can complement defects in yeast autophagy caused by loss of Apg6 and can also stimulate autophagy when overexpressed in mammalian cells (3). Mammalian Beclin-1 was originally isolated in a yeast two-hybrid screen for Bcl-2 interacting proteins and has been shown to interact with Bcl-2 and Bcl-xL, but not with Bax or Bak (4). While Beclin-1 is generally ubiquitously expressed, research studies have shown it is monoallelically deleted in 40-75% of sporadic human breast and ovarian cancers (5). Beclin-1 is localized within cytoplasmic structures including the mitochondria, although overexpression of Beclin-1 reveals some nuclear staining and CRM1-dependent nuclear export (6). Investigators have demonstrated that Beclin-1-/- mice die early in embryogenesis and Beclin-1-/+ mice have a high incidence of spontaneous tumors. Stem cells from the null mice demonstrate an altered autophagic response, although responses to apoptosis appeared normal (7). Researchers have also found that overexpression of Beclin-1 in virally infected neurons in vivo resulted in significant protection against Sindbis virus-induced disease and neuronal apoptosis (4).
Several studies have indicated that autophagy is enhanced by phosphorylation of Beclin-1 at Ser90 triggered by stress-responsive kinases including MAPKAPK-2/MAPKAPK-3, DAPK3, and CaMKII (8-10).
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