Western blot analysis of extracts from NIH/3T3 and 3T3-L1 cells using Adiponectin (C45B10) 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
Adiponectin (C45B10) Rabbit mAb detects endogenous levels of total adiponectin protein monomer. It will not detect higher molecular weight forms of adiponectin.Species Reactivity:
Human, Mouse, Rat
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to human adiponectin.
Adiponectin, also termed AdipoQ, Acrp30, apM1 and GBP28, is an adipokine expressed exclusively in brown and white adipocytes (1). It is secreted into the blood and exists in three major forms: a low molecular weight trimer, a medium molecular weight hexamer and a high molecular weight multimer (1). Adiponectin levels are decreased in obese and insulin-resistant mice and humans (2), suggesting that this adipokine is critical to maintain insulin sensitivity. Adiponectin stimulates the phosphorylation of AMPKα at Thr172 and activates AMPK in skeletal muscle (3). It also stimulates glucose uptake in myocytes (3). The block of AMPK activation by a dominant-negative AMPKα2 isoform inhibits the effect of adiponectin on glucose uptake, indicating that adiponectin stimulates glucose uptake and increases insulin sensitivity through its action on AMPK (3). Adiponectin mutants that are not able to form oligomers larger than trimers have no effect on the AMPK pathway (4). Mutations that render adiponectin unable to form high molecular weight multimers are associated with human diabetes (4), indicating the importance of multimerization for adiponectin activity.
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Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.