Western blot analysis of extracts from various cell lines using CtIP (D76F7) Rabbit mAb.
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
CtIP (D76F7) Rabbit mAb recognizes endogenous levels of total CtIP protein.Species Reactivity:
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human CtIP protein.
DNA damage checkpoints are critical for regulated repair of damaged DNA and genome maintenance. CtIP/RBBP8 (CtBP-interacting protein), initially characterized as a binding partner for the trancription factor CtBP, has emerged as a regulator of both cell cycle progression and repair of DNA double strand breaks (DSB). Along with the DSB-sensing MRN complex (MRE11-RAD50-NBS1), CtIP functions in the generation of single stranded DNA at DSBs, a process required for signaling to DNA repair machinery (reviewed in 1). CtIP is thought to be critical in the transition between sensing of DSBs and repair by homologous recombination (HR) (2,3).
In addition to HR, DSBs can also be repaired through nonhomologous end joining (NHEJ), and CtIP has been shown to have a role in signaling to the NHEJ pathway independently of its function in DSB end resection (4).
CtIP is also involved in cellular tolerence of topoisomerase inhibitors camptothecin and etoposide, which are used to treat cancer through their ability to introduce DSBs in cycling cells (5).
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