Western blot analysis of extracts from various cell lines using Alix (E4T7U) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Western blot analysis of extracts from HCT 116 and HCT 116 Alix knockout (-/-) cells using Alix (E4T7U) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (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 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
Alix (E4T7U) Rabbit mAb recognizes endogenous levels of total Alix protein.Species Reactivity:
Human, Mouse, Rat
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Arg475 of human Alix protein.
Alix, a phylogenetically conserved cytosolic scaffold protein, contains an N-terminal Bro1 domain, a coiled-coil region and a C-terminal proline-rich domain (1,2). Originally identified as an ALG-2 (apoptosis-linked gene 2)-interacting protein involved in programmed cell death (3,4), Alix also regulates many other cellular processes, such as endocytic membrane trafficking and cell adhesion through interactions with ESCRT (endosomal sorting complex required for transport) proteins, endophilins, and CIN85 (Cbl-interacting protein of 85 kDa) (5,6).
Alix has also been shown to be involved in the biogenesis of exosomes, small secreted vesicles that contribute to cell signaling (7, 8). In addition, Alix interacts with Atg12-Atg3 to promote autophagy and endosomal fusion (9)
Explore pathways + proteins related to this product.
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