Western blot analysis of extracts from various cell lines using Atg101 (E1Z4W) Rabbit mAb.
Western blot analysis of extracts from 293 cells, mock transfected (-) or transfected with a construct expressing Myc/DDK-tagged full-length human Atg101 protein (hAtg101-Myc/DDK; +), using Atg101 (E1Z4W) Rabbit mAb (upper) and 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, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 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.
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 June 2016
Protocol Id: 263
Atg101 (E1Z4W) Rabbit mAb recognizes endogenous levels of total Atg101 protein.Species Reactivity:
Human, Mouse, Rat, Monkey
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Val177 of human Atg101 protein.
Atg101 was discovered as a binding protein for Atg13, a component of the ULK1 serine-threonine kinase complex required for autophagy (1-3). Autophagy is a catabolic process for the autophagosomic-lysosomal degradation of bulk cytoplasmic contents (4,5). It is generally activated by conditions of nutrient deprivation, but is also associated with a number of physiological processes including development, differentiation, neurodegeneration, infection, and cancer (6). The molecular machinery of autophagy was largely discovered in yeast and is directed by a number of autophagy-related (Atg) genes. The ULK1 complex includes both Atg13 and FIP200 and is required for starvation-induced autophagy (7-9). Interaction between Atg101 and Atg13 can be important for the stability and basal phosphorylation of Atg13 and ULK1 (1,2).
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