Western blot analysis of extracts from various cell lines using Pontin/RUVBL1 Antibody.
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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
Pontin/RUVBL1 Antibody recognizes endogenous levels of total Pontin/RUVBL1 protein.Species Reactivity:
Human, Mouse, Rat, Hamster, Monkey
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human Pontin/RUVBL1 protein. Antibodies are purified by protein A and peptide affinity chromatography.
Reptin/RuvBL2 and Pontin/RuvBL1 are closely related members of the AAA+ (ATPase associated with diverse cellular activities) superfamily of proteins, and are putatively homologous to bacterial RuvB proteins that drive branch migration of Holliday junctions (1). Reptin and Pontin function together as essential components of chromatin remodeling and modification complexes, such as INO80, TIP60, SRCAP, and Uri1, which play key roles in regulating gene transcription (1,2). In their capacity as essential transcriptional co-regulators, Reptin and Pontin have both been implicated in oncogenic transformations, including those driven by c-Myc, β-catenin, and E1A (2-7).
A number of research studies have reported elevated levels of Pontin in selected cancer subtypes, including hepatocellular carcinoma and colon cancer (8-10). Interestingly, Pontin was reported to potentiate the TCF/LEF1-β-catenin transcriptional complex, whereas Reptin was antagonistic to the same complex, highlighting their related, but non-redundant, function (5). Research studies also suggest a functional role for Pontin and Reptin in the DNA damage response. Both proteins are phosphorylated by ATM and Rad3-related following DNA damage (11), while Pontin is reportedly required for TIP60 activity during the DNA damage response (12).
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