Western blot analysis of extracts from HT-1376 cells, untreated or pervanadate-treated ( 1 mM, 20 minutes), using Phospho-p73 (Tyr99) Antibody.
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
Phospho-p73 (Tyr99) Antibody detects endogenous levels of p73 only when phosphorylated at tyrosine 99. This antibody cross-reacts with p63 when phosphorylated at tyrosine 149.Species Reactivity:
HumanSpecies predicted to react based on 100% sequence homology:
Mouse, Rat, Monkey
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Tyr99 of human p73. Antibodies are purified by protein A and peptide affinity chromatography.
The p53 family member, p73, exists in multiple isoforms/splice variants and can induce apoptosis and cell cycle arrest in response to DNA damage via its activity as a transcription regulator (1-3). Upon DNA damage, p73 is phosphorylated at Tyr99 by c-Abl, causing translocation to the nuclear matrix (4). DNA damage-induced acetylation of p73 at Lys321 by the acetyltransferase p300 has also been reported to enhance transcription of genes including that of p53AIP1 (5). Another report, however, indicates that p300 does not acetylate full-length p73 in vivo (6).
While the sequences surrounding p73 Tyr99 and p63 Tyr149 are very similar, only p73 co-localizes with c-Abl following gamma irradiation, suggesting that p63 is not a c-Abl substrate (7).
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