Western blot analysis of extracts from human and mouse lung tissue using RAGE Antibody (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).
|MW (kDa)||58, 52, 48|
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
RAGE Antibody detects endogenous levels of total RAGE protein. It detects isoform 1 of human RAGE, and is predicted to detect multiple RAGE isoforms in mouse, including xRAGE, mRAGE, and sRAGE.Species Reactivity:
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ala23 of human RAGE protein. Antibodies are purified by peptide affinity chromatography.
The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin (Ig) superfamily. It can be expressed as full-length, membrane-bound RAGE isoform 1 or as a secreted sRAGE protein that lacks a transmembrane domain (1). RAGE is detected during early developmental stages and in the lung under normal physiological conditions (2), and it is upregulated at sites of inflammation (3). Advanced glycation end products (AGEs) and a variety of other ligands interact with this receptor (1). Ligand binding activates full-length RAGE and initiates downstream signaling pathways that include activation of NF-κB, which leads to production of pro-inflammatory cytokines and inflammation (4). Activation of these pathways has been implicated in various disease states including Alzheimer's disease, diabetes, arthritis, and atherosclerosis (4). Soluble RAGE can competitively bind RAGE ligands in the extracellular environment, which prevents ligand interaction with full-length RAGE at the cell surface (1).
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