Western blot analysis of extracts from HL-60 cells, untreated or treated with retinoic acid for the indicated times (RA; 1 μM), using APR3 Antibody.Learn more about how we get our images.
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with an expression construct encoding tagged APR3 (+), using APR3 Antibody. The construct was kindly provided by Drs. Timothy Peterson and Erin O'Shea, Harvard University, Boston MA.Learn more about how we get our images.
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
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.
APR3 Antibody recognizes endogenous levels of total APR3 protein.
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro29 of human APR3 protein (isoform a). Antibodies are purified by protein A and peptide affinity chromatography.
Apoptosis related protein 3 (APR3), also known as C2orf28, is a membrane protein identified in HL-60 cells treated with all-trans retinoic acid (ATRA) and was later found to be induced by ATRA in other sensitive cell lines (1,2). APR3 is also up-regulated by NFAT and NF-κB activities (3). Regulation of APR3 by ATRA suggests a role in cell differentiation, but the mechanism of action is still unclear. Overexpression of APR3 can inhibit proliferation by inducing G1/S cell cycle arrest and decreasing expression of cyclin D1 (2). APR3 has also been shown to interact with NELL-1 to regulate osteoblast differentiation (4).
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