Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a mouse HIPK2 construct (+), using HIPK2 Antibody.
Western blot analysis of extracts from Raji cells, mouse brain, and PC-12 cells using HIPK2 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
HIPK2 Antibody detects endogenous levels of total HIPK2 protein.Species Reactivity:
Human, Mouse, Rat
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Gln1045 of human HIPK2 protein. Antibodies were purified by protein A and peptide affinity chromatography.
Members of the homeodomain-interacting protein kinase (HIPK1-4) family of serine/threonine kinases regulate gene transcription with effects on cell proliferation, differentiation, and apoptosis (1-3). HIPK1-3 are nuclear proteins that were originally described as co-repressors for homeobox transcription factors (1). HIPK proteins can interact with and/or phosphorylate many transcriptional regulators (4).
HIPK2 activated in response to DNA damage, including UV radiation and chemotherapeutic drugs, phosphorylates p53 at Ser46 to promote the transcription of pro-apoptotic p53 target genes (5-7). In addition, HIPK2 interacts with a number of transcription factors that control developmental processes, tumor suppression and apoptosis (4). The kinase is regulated by both sumoylation (8) and ubiquitination (9,10). Ubiquitination and subsequent degradation of HIPK2 is inhibited by DNA damaging agents. Caspase-dependent cleavage of HIPK2 removes the inhibitory domain and results in enhanced HIPK2 activity (11).
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