Western blot analysis of extracts from various cell lines using Ambra1 Antibody.
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected (+) with a construct expressing Myc/DDK-tagged full-length human Ambra1 (hAmbra1-Myc/DDK) using Ambra1 Antibody (upper) or Myc-Tag (71D10) Rabbit mAb #2278 (lower).
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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
Ambra1 Antibody recognizes endogenous levels of total Ambra1 protein. A band of unknown origin is observed at 75 kDa in some cell lines.Species Reactivity:
Human, Mouse, Rat
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Leu220 of human Ambra1 protein. Antibodies are purified by protein A and peptide affinity chromatography.
Activating molecule in Beclin1-regulated autophagy (Ambra1) is a WD40-containing protein expressed during neurodevelopment that is required for neural tube development and autophagy (1). Several studies have identified interactions between Ambra1 with regulators of autophagy and apoptosis (reviewed in 2). Ambra1 was originally found to interact with Beclin-1, a key protein responsible for activating the class III PI3K Vps34 (1). Further studies showed that Ambra1 tethers the Beclin-1-Vps34 complex to the cytoskeletal network through dynein light chains and that during autophagy ULK1 phosphorylates Ambra1, resulting in disassociation with dynein and translocation of the Beclin-Vps34 complex to the endoplasmic reticulum to initiate autophagosome formation (3,4). In addition, it has been found that Ambra1 binds to mitochondrial Bcl-2 and that this interaction is regulated by either apoptosis or autophagy (5,6). Ambra1 also interacts with Parkin, an E3 ubiquitin ligase important for mitophagy, a selective autophagic process of mitochondrial clearance (7,8).
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