Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Myc/DDK-tagged full-length human atlastin-1 (hAlastin-1-Myc/DDK; +), atlastin-2 (hAlastin-2-Myc/DDK; +) or atlastin-3 (hAlastin-3-Myc/DDK; +) protein, using Atlastin-1 (D2E6) Rabbit mAb (upper) or Myc-Tag (71D10) Rabbit mAb #2278 (lower).
Western blot analysis of extracts from human, mouse and rat brain using Atlastin-1 (D2E6) Rabbit mAb.
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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 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
Atlastin-1 (D2E6) Rabbit mAb recognizes endogenous levels of total atlastin-1 protein. This antibody does not cross-react with atlastin-2 or atlastin-3. This antibody may cross-react with a protein of unknown origin at 100 kDa.Species Reactivity:
Human, Mouse, Rat
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human atlastin-1 protein.
Atlastin proteins are highly conserved members of the dynamin superfamily of membrane GTPases that are involved in the formation of vesicles for the endocytotic and secretory processes (1). Atlastins are required to establish and maintain the morphology of the tubular endoplasmic reticulum (ER) and are therefore important in ER function (2). GTP hydrolysis and dimerization are required for atlastin-dependent ER membrane fusion (3).
Atlastin GTPase 1 (ATL1) is primarily expressed in brain, while the related atlastin 2 and atlastin 3 proteins are ubiquitously expressed (4). Mutations in the atlastin 1 gene SPG3A and the ER defects that result are thought to cause one form of hereditary spastic paraplegia (HSP), a group of heterogeneous neurological disorders characterized by severe progressive spasticity of the lower limbs (5,6).
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