Western blot analysis of NIH/3T3 cells using Phospho-HDAC4 (Ser246)/HDAC5 (Ser259)/HDAC7 (Ser155) (D27B5)
Rabbit mAb #3443, HDAC4 (D8T3Q) Rabbit mAb #15164, HDAC5 (D1J7V) Rabbit mAb #20458, and HDAC7 (D4E1L) Rabbit mAb #33418. To show the phospho-specificity of the antibody, the blot was mock treated (-) or treated (+) with calf intestinal phosphatase (CIP) and lambda phosphatase. As expected, signal from the Phospho-HDAC4 (Ser246)/HDAC5 (Ser259)/HDAC7 (Ser155) (D27B5) Rabbit mAb is lost after treatment of the blot with phosphatase.
|MW (kDa)||140, 124|
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
Phospho-HDAC4 (Ser246)/HDAC5 (Ser259)/HDAC7 (Ser155) (D27B5) Rabbit mAb detects endogenous levels of HDAC4, HDAC5 and HDAC7 proteins only when phosphorylated on Ser246, Ser259 and Ser155, respectively.Species Reactivity:
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to Ser155 of human HDAC7 protein.
Acetylation of the histone tail causes chromatin to adopt an "open" conformation, allowing increased accessibility of transcription factors to DNA. The identification of histone acetyltransferases (HATs) and their large multiprotein complexes has yielded important insights into how these enzymes regulate transcription (1,2). HAT complexes interact with sequence-specific activator proteins to target specific genes. In addition to histones, HATs can acetylate nonhistone proteins, suggesting multiple roles for these enzymes (3). In contrast, histone deacetylation promotes a "closed" chromatin conformation and typically leads to repression of gene activity (4). Mammalian histone deacetylases can be divided into three classes on the basis of their similarity to various yeast deacetylases (5). Class I proteins (HDACs 1, 2, 3, and 8) are related to the yeast Rpd3-like proteins, those in class II (HDACs 4, 5, 6, 7, 9, and 10) are related to yeast Hda1-like proteins, and class III proteins are related to the yeast protein Sir2. Inhibitors of HDAC activity are now being explored as potential therapeutic cancer agents (6,7).
Histone deacetylases (HDACs) interact with an increasing number of transcription factors, including myocyte enhancer factor 2 (MEF2), to negatively regulate gene expression. HDACs are regulated in part by shuttling between the nucleus and cytoplasm, where export to the cytoplasm facilitates gene activation by removing HDACs from their target genes (8,9). The cytoplasmic export is facilitated by 14-3-3 proteins, which bind to specific phospho-serine residues on the HDAC proteins (8,9). These phospho-serine 14-3-3 binding modules are highly conserved between HDAC proteins, allowing for their collective regulation in response to specific cell stimuli. For example, the highly conserved HDAC 4 Ser246, HDAC 5 Ser259 and HDAC 7 Ser155 residues are all phosphorylated by CAMK and PKD kinases in response to multiple cell stimuli, including VEGF-induced angiogenesis in endothelial cells, B cell and T cell activation, and differentiation of myoblasts into muscle fiber (10-14).
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