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78672
Ubiquityl-Histone H2A.Z (Lys120/Lys121) (E3J7J) Rabbit mAb
Primary Antibodies
Monoclonal Antibody

Ubiquityl-Histone H2A.Z (Lys120/Lys121) (E3J7J) Rabbit mAb #78672

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  1. WB
Western Blotting Image 1: Ubiquityl-Histone H2A.Z (Lys120/Lys121) (E3J7J) Rabbit mAb

Western blot analysis of extracts from various cell lines using Ubiquityl-Histone H2A.Z (Lys120/Lys121) (E3J7J) Rabbit mAb (upper) and H2A.Z (E9M5G) Rabit mAb #50722 (lower).

To Purchase # 78672S
Product # Size Price
78672S
100 µl $ 312

Supporting Data

REACTIVITY H M R Mk
SENSITIVITY Endogenous
MW (kDa) 23
Source/Isotype Rabbit IgG

Application Key:

  • W-Western
  • IP-Immunoprecipitation
  • IHC-Immunohistochemistry
  • ChIP-Chromatin Immunoprecipitation
  • IF-Immunofluorescence
  • F-Flow Cytometry
  • E-P-ELISA-Peptide

Species Cross-Reactivity Key:

  • H-Human
  • M-Mouse
  • R-Rat
  • Hm-Hamster
  • Mk-Monkey
  • 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
  • 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. Prestained Protein Marker, Broad Range (11-190 kDa): (#13953).
  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 (#13953, 5 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

Ubiquityl-Histone H2A.Z (Lys120/Lys121) (E3J7J) Rabbit mAb recognizes endogenous levels of histone H2A.Z protein when ubiquitylated at Lys120 and/or Lys121. This antibody shows very weak cross-reactivity with histone H2A ubiquitylated on Lys118 and Lys119. This antibody does not cross-react with other ubiquitylated proteins or free ubiquitin.

Species Reactivity:

Human, Mouse, Rat, Monkey

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding ubiquitylated Lys121 of human histone H2A.Z protein.

Background

Modulation of chromatin structure plays a critical role in the regulation of transcription in eukaryotes. The nucleosome, made up of four core histone proteins (H2A, H2B, H3 and H4), is the primary building block of chromatin. In addition to the growing number of post-translational histone modifications regulating chromatin structure, cells can also exchange canonical histones with variant histones that can directly or indirectly modulate chromatin structure (1). There are five major variants of histone H2A: canonical H2A (most abundant), H2A.X, MacroH2A, H2ABbd and H2A.Z (2). Histone H2A.Z, the most conserved variant across species, functions as both a positive and negative regulator of transcription and is important for chromosome stability (2). Several homologous protein complexes, such as SWR-C (S. cerevisiae), TIP60 (D. melanogaster) and SRCAP (mammals), have been shown to catalyze the ATP-dependent exchange of H2A.Z for H2A in the nucleosome (3,4,5). This exchange of histone H2A variants changes histone-histone interactions in the nucleosome core and alters an acidic patch on the surface of the nucleosome, resulting in changes in nucleosome stability and binding of non-histone proteins such as HP1α (6,7).

H2A.Z is a histone H2A variant protein that is critical for proper regulation of gene expression. H2A.Z is localized throughout the genome, but appears to be most concentrated at the promoters and enhancers of active genes (8). Acetylation of histone H2A.Z at promoters and enhancers confers nucleosome destabilization and open chromatin confirmation, facilitating transcriptional activation (9-11). While the bulk of histone H2A.Z appears to be excluded from constitutive heterochromatin, histone H2A.Z is found in various forms of facultative heterochromatin, including the inactive X chromosome and transcriptionally poised bivalent gene promoters (8,12). In these heterochromatic regions of the genome, H2A.Z is mono-ubiquitylated on Lys120 and Lys121 by the Ring1B ubiquitin ligase found in the Polycomb Repressor Complex 1 (PRC1) (8,12). Mono-methylation of H2A.Z on Lys120 and Lys121 facilitates repression of gene expression by inhibiting the binding of the activating BRD4 protein and facilitating the recruitment of the Polycomb Repressor Complex 2 (PRC2), the latter of which methylates histone H3 on Lys27 and facilitates transcriptional repression (8).

  1. Jin, J. et al. (2005) Trends Biochem Sci 30, 680-7.
  2. Raisner, R.M. and Madhani, H.D. (2006) Curr Opin Genet Dev 16, 119-24.
  3. Mizuguchi, G. et al. (2004) Science 303, 343-8.
  4. Kusch, T. et al. (2004) Science 306, 2084-7.
  5. Ruhl, D.D. et al. (2006) Biochemistry 45, 5671-7.
  6. Suto, R.K. et al. (2000) Nat Struct Biol 7, 1121-4.
  7. Fan, J.Y. et al. (2004) Mol Cell 16, 655-61.
  8. Surface, L.E. et al. (2016) Cell Rep 14, 1142-55.
  9. Millar, C.B. et al. (2006) Genes Dev 20, 711-22.
  10. Ishibashi, T. et al. (2009) Biochemistry 48, 5007-17.
  11. Valdés-Mora, F. et al. (2012) Genome Res 22, 307-21.
  12. Sarcinella, E. et al. (2007) Mol Cell Biol 27, 6457-68.

Pathways & Proteins

Explore pathways + proteins related to this product.

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