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97272
Citrullinated Histone H3 (Arg17) (E4O3F) Rabbit mAb
Primary Antibodies
Monoclonal Antibody

Citrullinated Histone H3 (Arg17) (E4O3F) Rabbit mAb #97272

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  1. WB
Western Blotting Image 1: Citrullinated Histone H3 (Arg17) (E4O3F) Rabbit mAb
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing human PADI4 (hPADI4; +), and untreated (-) or treated (+) as indicated with: Locke’s Solution (30 min) and A23187 ionophore (5 μM, 30 min), using Citrullinated Histone H3 (Arg17) (E4O3F) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). Citrullination of histone H3 is induced by expression of PADI4, along with activation by Locke’s Solution and A23187 ionophore treatment, as expected.
Product Image 1: Citrullinated Histone H3 (Arg17) (E4O3F) Rabbit mAb
Peptide dot blot analysis demonstrating Citrullinated Histone H3 (Arg17) (E4O3F) Rabbit mAb specificity. Antibody binding to pre-coated histone H3 peptides is shown using Citrullinated Histone H3 (Arg17) (E4O3F) Rabbit mAb. Citrullinated Histone H3 (Arg17) (E4O3F) Rabbit mAb binds to histone H3 peptide when citrullinated at Arg17, and may also bind to histone H3 peptide when citrullinated at Arg26.
To Purchase # 97272
Cat. # Size Qty. Price
97272S
100 µl
$ 287

Supporting Data

REACTIVITY H
SENSITIVITY Endogenous
MW (kDa) 17
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
  • 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

Citrullinated Histone H3 (Arg17) (E4O3F) Rabbit mAb recognizes histone H3 citrullinated at residue Arg17. This antibody may also recognize histone H3 citrullinated at residue Arg26, but does not cross-react with any other known citrullinated or methylated arginine residues on histone H3. This antibody may detect a band of unknown identity at 38 kDa.

Species Reactivity:

Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to the amino terminus of histone H3 in which Arg17 is citrullinated.

Background

The nucleosome, made up of four core histone proteins (H2A, H2B, H3, and H4), is the primary building block of chromatin. Histone posttranslational modifications, such as methylation, acetylation, ubiquitination, and citrullination, influence chromatin accessibility and regulate gene expression (1). Histone citrullination is carried out by protein arginine deiminases (PADs), which convert positively charged arginine residues into neutral citrulline (2). Histone citrullination affects the binding of histone reader proteins to chromatin, and it is associated with both transcriptional activation and repression (3-5). Histone citrullination is also a major marker for NETosis, which is a programmed cell death mechanism carried out by neutrophils (6). De-regulation of NETosis is associated with multiple autoimmune diseases, including rheumatoid arthritis (RA) and lupus (7,8). Histone hypercitrullination via PAD4 may also play a role in the progression of various cancers, including multiple myeloma (MM), acute myeloid leukemia (AML), lung cancer, and hepatocellular carcinoma (HCC) (9-12).
  1. Shanmugam, M.K. et al. (2018) Oncotarget 9, 11414-11426.
  2. Fuhrmann, J. and Thompson, P.R. (2016) ACS Chem Biol 11, 654-68.
  3. Wang, Y. et al. (2004) Science 306, 279-83.
  4. Cuthbert, G.L. et al. (2004) Cell 118, 545-53.
  5. Zhang, X. et al. (2012) Proc Natl Acad Sci U S A 109, 13331-6.
  6. Hamam, H.J. and Palaniyar, N. (2019) Biomolecules 9, 369. doi: 10.3390/biom9080369.
  7. Corsiero, E. et al. (2016) Front Immunol 7, 485.
  8. Hakkim, A. et al. (2010) Proc Natl Acad Sci U S A 107, 9813-8.
  9. McNee, G. et al. (2017) Leukemia 31, 373-381.
  10. Song, G. et al. (2016) Oncotarget 7, 3144-57.
  11. Tanikawa, C. et al. (2012) Nat Commun 3, 676.
  12. Lu, M. et al. (2020) J Med Virol 92, 1221-1230.

Pathways & Proteins

Explore pathways + proteins related to this product.

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For Research Use Only. Not For Use In Diagnostic Procedures.
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