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3443
Phospho-HDAC4 (Ser246)/HDAC5 (Ser259)/HDAC7 (Ser155) (D27B5) Rabbit mAb
Primary Antibodies
Monoclonal Antibody
R
Recombinant

Phospho-HDAC4 (Ser246)/HDAC5 (Ser259)/HDAC7 (Ser155) (D27B5) Rabbit mAb #3443

Citations (57)
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  1. WB
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.
To Purchase # 3443
Cat. # Size Qty. Price
3443T
20 µl
3443S
100 µl

Supporting Data

REACTIVITY H M
SENSITIVITY Endogenous
MW (kDa) 140, 124
Source/Isotype Rabbit IgG

Application Key:

  • WB-Western Blot
  • IP-Immunoprecipitation
  • IHC-Immunohistochemistry
  • ChIP-Chromatin Immunoprecipitation
  • C&R-CUT&RUN
  • C&T-CUT&Tag
  • DB-Dot Blot
  • eCLIP-eCLIP
  • IF-Immunofluorescence
  • F-Flow Cytometry

Species Cross-Reactivity Key:

  • H-Human
  • M-Mouse
  • R-Rat
  • Hm-Hamster
  • Mk-Monkey
  • Vir-Virus
  • Mi-Mink
  • C-Chicken
  • Dm-D. melanogaster
  • X-Xenopus
  • Z-Zebrafish
  • B-Bovine
  • Dg-Dog
  • Pg-Pig
  • Sc-S. cerevisiae
  • Ce-C. elegans
  • Hr-Horse
  • GP-Guinea Pig
  • Rab-Rabbit
  • All-All Species Expected

Product Usage Information

Application Dilution
Western Blotting 1:1000

Storage

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.

Protocol

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Western Blotting Protocol

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 product webpage for recommended antibody dilution.

A. Solutions and Reagents

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.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 10X Tris Buffered Saline (TBS): (#12498) To prepare 1 L 1X TBS: add 100 ml 10X to 900 ml dH2O, mix.
  3. 1X SDS Sample Buffer: Blue Loading Pack (#7722) or Red Loading Pack (#7723) Prepare fresh 3X reducing loading buffer by adding 1/10 volume 30X DTT to 1 volume of 3X SDS loading buffer. Dilute to 1X with dH2O.
  4. 10X Tris-Glycine SDS Running Buffer: (#4050) To prepare 1 L 1X running buffer: add 100 ml 10X running buffer to 900 ml dH2O, mix.
  5. 10X Tris-Glycine Transfer Buffer: (#12539) To prepare 1 L 1X Transfer Buffer: add 100 ml 10X Transfer Buffer to 200 ml methanol + 700 ml dH2O, mix.
  6. 10X Tris Buffered Saline with Tween® 20 (TBST): (#9997) To prepare 1 L 1X TBST: add 100 ml 10X TBST to 900 ml dH2O, mix.
  7. Nonfat Dry Milk: (#9999).
  8. Blocking Buffer: 1X TBST with 5% w/v nonfat dry milk; for 150 ml, add 7.5 g nonfat dry milk to 150 ml 1X TBST and mix well.
  9. Wash Buffer: (#9997) 1X TBST.
  10. Bovine Serum Albumin (BSA): (#9998).
  11. Primary Antibody Dilution Buffer: 1X TBST with 5% BSA; for 20 ml, add 1.0 g BSA to 20 ml 1X TBST and mix well.
  12. Biotinylated Protein Ladder Detection Pack: (#7727).
  13. Blue Prestained Protein Marker, Broad Range (11-250 kDa): (#59329).
  14. Blotting Membrane and Paper: (#12369) This protocol has been optimized for nitrocellulose membranes. Pore size 0.2 µm is generally recommended.
  15. Secondary Antibody Conjugated to HRP: Anti-rabbit IgG, HRP-linked Antibody (#7074).
  16. Detection Reagent: SignalFire™ ECL Reagent (#6883).

B. Protein Blotting

A general protocol for sample preparation.

  1. Treat cells by adding fresh media containing regulator for desired time.
  2. Aspirate media from cultures; wash cells with 1X PBS; aspirate.
  3. Lyse cells by adding 1X SDS sample buffer (100 µl per well of 6-well plate or 500 µl for a 10 cm diameter plate). Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Keep on ice.
  4. Sonicate for 10–15 sec to complete cell lysis and shear DNA (to reduce sample viscosity).
  5. Heat a 20 µl sample to 95–100°C for 5 min; cool on ice.
  6. Microcentrifuge for 5 min.
  7. Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).

    NOTE: Loading of prestained molecular weight markers (#59329, 10 µl/lane) to verify electrotransfer and biotinylated protein ladder (#7727, 10 µl/lane) to determine molecular weights are recommended.

  8. Electrotransfer to nitrocellulose membrane (#12369).

C. Membrane Blocking and Antibody Incubations

NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.

I. Membrane Blocking

  1. (Optional) After transfer, wash nitrocellulose membrane with 25 ml TBS for 5 min at room temperature.
  2. Incubate membrane in 25 ml of blocking buffer for 1 hr at room temperature.
  3. Wash three times for 5 min each with 15 ml of TBST.

II. Primary Antibody Incubation

  1. Incubate membrane and primary antibody (at the appropriate dilution and diluent as recommended in the product webpage) in 10 ml primary antibody dilution buffer with gentle agitation overnight at 4°C.
  2. Wash three times for 5 min each with 15 ml of TBST.
  3. Incubate membrane with Anti-rabbit IgG, HRP-linked Antibody (#7074 at 1:2000) and anti-biotin, HRP-linked Antibody (#7075 at 1:1000–1:3000) to detect biotinylated protein markers in 10 ml of blocking buffer with gentle agitation for 1 hr at room temperature.
  4. Wash three times for 5 min each with 15 ml of TBST.
  5. Proceed with detection (Section D).

D. Detection of Proteins

Directions for Use:

  1. Wash membrane-bound HRP (antibody conjugate) three times for 5 minutes in TBST.
  2. Prepare 1X SignalFire™ ECL Reagent (#6883) by diluting one part 2X Reagent A and one part 2X Reagent B (e.g. for 10 ml, add 5 ml Reagent A and 5 ml Reagent B). Mix well.
  3. Incubate substrate with membrane for 1 minute, remove excess solution (membrane remains wet), wrap in plastic and expose to X-ray film.

* Avoid repeated exposure to skin.

posted June 2005

revised June 2020

Protocol Id: 10

Specificity / Sensitivity

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:

Human, Mouse

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to Ser155 of human HDAC7 protein.

Background

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).

  1. Marmorstein, R. (2001) Cell Mol Life Sci 58, 693-703.
  2. Gregory, P.D. et al. (2001) Exp Cell Res 265, 195-202.
  3. Liu, Y. et al. (2000) Mol Cell Biol 20, 5540-53.
  4. Cress, W.D. and Seto, E. (2000) J Cell Physiol 184, 1-16.
  5. Gray, S.G. and Ekström, T.J. (2001) Exp Cell Res 262, 75-83.
  6. Thiagalingam, S. et al. (2003) Ann. N.Y. Acad. Sci. 983, 84-100.
  7. Vigushin, D.M. and Coombes, R.C. (2004) Curr Cancer Drug Targets 4, 205-18.
  8. Grozinger, C.M. and Schreiber, S.L. (2000) Proc Natl Acad Sci U S A 97, 7835-40.
  9. Wang, A.H. et al. (2000) Mol Cell Biol 20, 6904-12.
  10. Ha, C.H. et al. (2008) J Biol Chem 283, 14590-9.
  11. Wang, S. et al. (2008) Proc Natl Acad Sci U S A 105, 7738-43.
  12. Matthews, S.A. et al. (2006) Mol Cell Biol 26, 1569-77.
  13. Parra, M. et al. (2005) J Biol Chem 280, 13762-70.
  14. McKinsey, T.A. et al. (2000) Nature 408, 106-11.

Pathways

Explore pathways related to this product.

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