Western blot analysis of extracts from 293T cells, untreated or treated with λ and calf intestinal phosphatases, using Phospho-ATRIP (Ser224) Antibody (upper) or ATRIP Antibody #2737 (lower).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.
Phospho-ATRIP (Ser224) Antibody detects endogenous levels of ATRIP protein only when phosphorylated at Ser224.
Polyclonal antibodies are produced by immunizing animals with a synthetic phoshopeptide corresponding to residues surrounding Ser224 of the human ATRIP protein.
In response to genomic stress, the ATR interacting protein (ATRIP) binds and is phosphorylated by the DNA damage-and checkpoint-activated kinase ATR (ataxia-telangiectasia mutated and rad3-related). Both ATR and ATRIP are integral for checkpoint signaling and are critical in the DNA repair response (1-3). Direct interaction between ATRIP and replication protein A (RPA) at RPA-coated, single-stranded DNA results in the recruitment of phosphorylated ATR/ATRIP to stalled replication forks and sites of DNA damage (3). ATR/ATRIP coordinate DNA repair and cell cycle progression in conjunction with key regulatory proteins, such as Rad17 and the 9-1-1 complex (4). ATR associated with ATRIP can also be stimulated by topoisomerase II binding protein (TOPBP1), suggesting that ATRIP may regulate both ATR localization and activity (5).
Cyclin dependent kinase 2 (CDK2) may participate in the regulation of DNA damage response and cell cycle control through phosphorylation of ATRIP at Ser224 (6).
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