Anti-Histone H4 (citrulline 3)

Species Reactivity	Key Applications	Host	Format	Antibody Type
H, M	ChIP, ICC, IH(P), WB	Rb	Affinity Purified	Polyclonal Antibody

Species Reactivity

Key Applications

Host

Format

Antibody Type

H, M

ChIP, ICC, IH(P), WB

Affinity Purified

Polyclonal Antibody

Description
Catalogue Number	07-596
Brand Family	Upstate
Trade Name	Upstate
Description	Anti-Histone H4 (citrulline 3) Antibody
Alternate Names	Cit-H4R3 H4Cit3 Histone H4, Arg3 citrullinated
Background Information	Histone H4 (UniProt P62805; also known as H4 histone family member A, Histone 1 H4a) is encoded by the HIST1H4A (also known as H4/A, H4FA) gene (Gene ID 8359) in human. Histone H4 is one of the main histone proteins that contribute to the chromatin structure in both prokaryotic and eukaryotic cells. Histone H4 can undergo several different types of epigenetic modifications that influence cellular processes, including methylation and acetylation on lysine and arginine residues, phosphorylation on serine and threonine residues, as well as conversion Arg3 to a citrulline residue by the peptidylarginine deiminase PAD4-catalyzed deimination reaction, also known as citrullination). Histone H4 Arg3 citrullination (H4Cit3) weakens histone-DNA interaction as a result of a conformational change and a reduced positive charge at the histone H4 N terminal end. Bacterial infection is known to stimulate H4Cit3 in neutrophils, forming highly decondensed extracellular chromatin structure NET (neutrophil extracellular trap) that could capture and kill microorganisms. On the other hand, citrullinated proteins are recognized as non-self proteins and subsequently activate immune systems. A functional haplotype of the PADI4 gene has been shown to be associated with the pathogenesis of rheumatoid arthritis through induction of the antigenicity of proteins. H4Cit3 is induced in response to DNA damage through the p53-PADI4 pathway, resulting in upregulated H4Cit3 immunoreactivity localized around fragmented nuclei in apoptotic cells. In addition, H4Cit3 level is negatively correlated with p53 protein expression and tumour size in non-small cell lung cancer (NSCLC) tissues, indicating a crucial role of H4Cit3 in carcinogenesis.

Description

Catalogue Number

07-596

Brand Family

Upstate

Trade Name

Upstate

Description

Anti-Histone H4 (citrulline 3) Antibody

Alternate Names

Cit-H4R3
H4Cit3
Histone H4, Arg3 citrullinated

Background Information

Histone H4 (UniProt P62805; also known as H4 histone family member A, Histone 1 H4a) is encoded by the HIST1H4A (also known as H4/A, H4FA) gene (Gene ID 8359) in human. Histone H4 is one of the main histone proteins that contribute to the chromatin structure in both prokaryotic and eukaryotic cells. Histone H4 can undergo several different types of epigenetic modifications that influence cellular processes, including methylation and acetylation on lysine and arginine residues, phosphorylation on serine and threonine residues, as well as conversion Arg3 to a citrulline residue by the peptidylarginine deiminase PAD4-catalyzed deimination reaction, also known as citrullination). Histone H4 Arg3 citrullination (H4Cit3) weakens histone-DNA interaction as a result of a conformational change and a reduced positive charge at the histone H4 N terminal end. Bacterial infection is known to stimulate H4Cit3 in neutrophils, forming highly decondensed extracellular chromatin structure NET (neutrophil extracellular trap) that could capture and kill microorganisms. On the other hand, citrullinated proteins are recognized as non-self proteins and subsequently activate immune systems. A functional haplotype of the PADI4 gene has been shown to be associated with the pathogenesis of rheumatoid arthritis through induction of the antigenicity of proteins. H4Cit3 is induced in response to DNA damage through the p53-PADI4 pathway, resulting in upregulated H4Cit3 immunoreactivity localized around fragmented nuclei in apoptotic cells. In addition, H4Cit3 level is negatively correlated with p53 protein expression and tumour size in non-small cell lung cancer (NSCLC) tissues, indicating a crucial role of H4Cit3 in carcinogenesis.

Product Information
Format	Affinity Purified
Presentation	Purified rabbit polyclonal antibody in buffer containing 0.1 M Tris-Glycine (pH 7.4), 150 mM NaCl with 0.05% sodium azide.

Product Information

Format

Affinity Purified

Presentation

Purified rabbit polyclonal antibody in buffer containing 0.1 M Tris-Glycine (pH 7.4), 150 mM NaCl with 0.05% sodium azide.

Applications
Application	Anti-Histone H4 (Citrulline 3) Antibody, Cat. No. 07-596, is a highly specific rabbit polyclonal antibody that targets histone H4 citrullination (H4Cit3) and has been tested in Chromatin Immunoprecipitation (ChIP), Immunocytochemistry, Immunohistochemistry, and Western Blotting.
Key Applications	Chromatin Immunoprecipitation (ChIP) Immunocytochemistry Immunohistochemistry (Paraffin) Western Blotting
Application Notes	Chromatin Immunoprecipitation (ChIP) Analysis: A representative lot (3 µL per ChIP) detected histone H4 Arg3 citrullination (H4Cit3) enrichment at the pS2 gene promoter in MCF-7 cells that had been treated with β-estradiol for one hour after a 3-day serum-starvation period. Immunocytochemistry Analysis: A 1:500 dilution detected from a representative lot detected enhanced histone H4 Arg3 citrullination in ionomycin-stimulated primary human neutrophils (Courtesy of Kerri Mowen, Ph.D., Padlock Therapeutics). Immunohistochemistry Analysis: A 1:250 dilution from a representative lot detected enhanced histone H4 Arg3 citrullination (H4Cit3) in 4% paraformaldehyde-fixed, decalcified, paraffin-embedded hind paw sections from wild-type, but not Pad4-knockout mice 5 days post immunization with arthritogenic serum from K/BxN mice (Courtesy of Kerri Mowen, Ph.D., Padlock Therapeutics). Chromatin Immunoprecipitation (ChIP) Analysis: A representative lot detected a time-dependent histone H4 Arg3 citrullination (H4Cit3) enrichment at the pS2 gene promoter in MCF-7 cells following estradiol treatment (Wang, Y., et al. (2004). Science. 306(5694):279-283). Immunocytochemistry Analysis: A representative lot detected enhanced nuclear H4/H2A Ar3 citrullination in HEK293T and U2OS cells upon exogenous PAD4 expression or DNA damage induction (Tanikawa, C., et al. (2012). Nat. Commun. 3:676). Immunocytochemistry Analysis: A representative lot detected Arg3-citrullinated histone H4 (H4Cit3) in 3.8% paraformaldehyde-fixed, 2% NP-40/1% Triton X-100-permeabilized casein-elicited peritoneal neutrophils cells from wild-type, but not Pad4-knockout mice (Hemmers, S., et al. (2011). PLoS One. 6(7):e22043). Immunocytochemistry Analysis: A representative lot detected Arg3-citrullinated histone H4 (H4Cit3) in 3.8% paraformaldehyde-fixed, 2% NP-40/1% Triton X-100-permeabilized infiltrating lung leukocytes isolated from wild-type, but not Pad4-knockout mice following influenza infection (Hemmers, S., et al. (2011). PLoS One. 6(7):e22043). Immunocytochemistry Analysis: A representative lot detected Arg3-citrullinated histone H4 (H4Cit3) in 4% paraformaldehyde-fixed, 1% NP-40/0.5% Triton X-100-permeabilized peripheral blood cells from wild-type, but not Pad4-knockout mice 5 days post immunization with arthritogenic serum from K/BxN mice (Rohrbach, A.S., et al. (2012). Arthritis Res. Ther. 14(3):R104). Immunocytochemistry Analysis: A representative lot detected Arg3-citrullinated histone H4 (H4Cit3) in synovial cells and extracellular traps (NETs) flushed out of the ankle joints of wild-type, but not Pad4-knockout mice 5 days post immunization with arthritogenic serum from K/BxN mice (Rohrbach, A.S., et al. (2012). Arthritis Res. Ther. 14(3):R104). Immunocytochemistry Analysis: A representative lot detected calcium ionophore-induced histone H4 (H4Cit3) Arg3 citrullination in 3.7% paraformaldehyde-fixed, 0.2% NP-40/0.1% Triton X-100-permeabilized HL-60 cells (Wang, Y., et al. (2004). Science. 306(5694):279-283). Immunohistochemistry Analysis: A representative lot detected gamma irradiation-induced nuclear H4/H2A Ar3 citrullination in thymus tissue sections from wild-type, but not Pad4-knockout mice (Tanikawa, C., et al. (2012). Nat. Commun. 3:676). Immunohistochemistry Analysis: A representative lot detected enhanced histone H4 Arg3 citrullination (H4Cit3) in 4% paraformaldehyde-fixed, decalcified, paraffin-embedded hind paw sections from wild-type, but not Pad4-knockout mice 10 days post immunization with arthritogenic serum from K/BxN mice (Rohrbach, A.S., et al. (2012). Arthritis Res. Ther. 14(3):R104). Western Blotting Analysis: A representative lot detected PAD4-catalyzed Arg3 citrullination of recombinant histone H4 and H2A, as well as DNA damage-induced cellular H4 and H2A Ar3 citrullination (Tanikawa, C., et al. (2012). Nat. Commun. 3:676). Western Blotting Analysis: A representative lot detected calcium ionophore-induced histone H4 Arg3 citrullination (H4Cit3) in HL-60 cells. Cellular PAD4 knockdown by shRNA treatment greatly diminished ionophor-induced H4Cit3, while antibody blocking with the immunogen peptide prevented target band detection (Wang, Y., et al. (2004). Science. 306(5694):279-283).

Applications

Application

Anti-Histone H4 (Citrulline 3) Antibody, Cat. No. 07-596, is a highly specific rabbit polyclonal antibody that targets histone H4 citrullination (H4Cit3) and has been tested in Chromatin Immunoprecipitation (ChIP), Immunocytochemistry, Immunohistochemistry, and Western Blotting.

Key Applications

Chromatin Immunoprecipitation (ChIP)
Immunocytochemistry
Immunohistochemistry (Paraffin)
Western Blotting

Application Notes

Chromatin Immunoprecipitation (ChIP) Analysis: A representative lot (3 µL per ChIP) detected histone H4 Arg3 citrullination (H4Cit3) enrichment at the pS2 gene promoter in MCF-7 cells that had been treated with β-estradiol for one hour after a 3-day serum-starvation period.

Immunocytochemistry Analysis: A 1:500 dilution detected from a representative lot detected enhanced histone H4 Arg3 citrullination in ionomycin-stimulated primary human neutrophils (Courtesy of Kerri Mowen, Ph.D., Padlock Therapeutics).

Immunohistochemistry Analysis: A 1:250 dilution from a representative lot detected enhanced histone H4 Arg3 citrullination (H4Cit3) in 4% paraformaldehyde-fixed, decalcified, paraffin-embedded hind paw sections from wild-type, but not Pad4-knockout mice 5 days post immunization with arthritogenic serum from K/BxN mice (Courtesy of Kerri Mowen, Ph.D., Padlock Therapeutics).

Chromatin Immunoprecipitation (ChIP) Analysis: A representative lot detected a time-dependent histone H4 Arg3 citrullination (H4Cit3) enrichment at the pS2 gene promoter in MCF-7 cells following estradiol treatment (Wang, Y., et al. (2004). Science. 306(5694):279-283).

Immunocytochemistry Analysis: A representative lot detected enhanced nuclear H4/H2A Ar3 citrullination in HEK293T and U2OS cells upon exogenous PAD4 expression or DNA damage induction (Tanikawa, C., et al. (2012). Nat. Commun. 3:676).

Immunocytochemistry Analysis: A representative lot detected Arg3-citrullinated histone H4 (H4Cit3) in 3.8% paraformaldehyde-fixed, 2% NP-40/1% Triton X-100-permeabilized casein-elicited peritoneal neutrophils cells from wild-type, but not Pad4-knockout mice (Hemmers, S., et al. (2011). PLoS One. 6(7):e22043).

Immunocytochemistry Analysis: A representative lot detected Arg3-citrullinated histone H4 (H4Cit3) in 3.8% paraformaldehyde-fixed, 2% NP-40/1% Triton X-100-permeabilized infiltrating lung leukocytes isolated from wild-type, but not Pad4-knockout mice following influenza infection (Hemmers, S., et al. (2011). PLoS One. 6(7):e22043).

Immunocytochemistry Analysis: A representative lot detected Arg3-citrullinated histone H4 (H4Cit3) in 4% paraformaldehyde-fixed, 1% NP-40/0.5% Triton X-100-permeabilized peripheral blood cells from wild-type, but not Pad4-knockout mice 5 days post immunization with arthritogenic serum from K/BxN mice (Rohrbach, A.S., et al. (2012). Arthritis Res. Ther. 14(3):R104).

Immunocytochemistry Analysis: A representative lot detected Arg3-citrullinated histone H4 (H4Cit3) in synovial cells and extracellular traps (NETs) flushed out of the ankle joints of wild-type, but not Pad4-knockout mice 5 days post immunization with arthritogenic serum from K/BxN mice (Rohrbach, A.S., et al. (2012). Arthritis Res. Ther. 14(3):R104).

Immunocytochemistry Analysis: A representative lot detected calcium ionophore-induced histone H4 (H4Cit3) Arg3 citrullination in 3.7% paraformaldehyde-fixed, 0.2% NP-40/0.1% Triton X-100-permeabilized HL-60 cells (Wang, Y., et al. (2004). Science. 306(5694):279-283).
Immunohistochemistry Analysis: A representative lot detected gamma irradiation-induced nuclear H4/H2A Ar3 citrullination in thymus tissue sections from wild-type, but not Pad4-knockout mice (Tanikawa, C., et al. (2012). Nat. Commun. 3:676).

Immunohistochemistry Analysis: A representative lot detected enhanced histone H4 Arg3 citrullination (H4Cit3) in 4% paraformaldehyde-fixed, decalcified, paraffin-embedded hind paw sections from wild-type, but not Pad4-knockout mice 10 days post immunization with arthritogenic serum from K/BxN mice (Rohrbach, A.S., et al. (2012). Arthritis Res. Ther. 14(3):R104).

Western Blotting Analysis: A representative lot detected PAD4-catalyzed Arg3 citrullination of recombinant histone H4 and H2A, as well as DNA damage-induced cellular H4 and H2A Ar3 citrullination (Tanikawa, C., et al. (2012). Nat. Commun. 3:676).

Western Blotting Analysis: A representative lot detected calcium ionophore-induced histone H4 Arg3 citrullination (H4Cit3) in HL-60 cells. Cellular PAD4 knockdown by shRNA treatment greatly diminished ionophor-induced H4Cit3, while antibody blocking with the immunogen peptide prevented target band detection (Wang, Y., et al. (2004). Science. 306(5694):279-283).

Biological Information
Immunogen	KLH-conjugated, synthetic peptide corresponding to amino acids 1-10 (SG[cit]GKGGKGL-C) of human histone H4 (Met1 removed), where [cit] denotes citrullinated Arg3. A C-terminal cysteine was added for conjugation purposes.
Epitope	N-terminus
Concentration	Please refer to lot specific datasheet.
Host	Rabbit
Specificity	This rabbit polyclonal antibody detected PAD4-catalyzed Arg3 citrullination of recombinant histone H4 and H2A, as well as calcium ionophore-induced histone H4 Arg3 citrullination (H4Cit3) in HL-60 cells. Cellular PAD4 (peptidyl arginine deiminase 4) knockdown greatly diminished ionophor-induced H4Cit3, while antibody blocking with the immunogen peptide prevented target band detection (Tanikawa, C., et al. (2012). Nat. Commun. 3:676; Wang, Y., et al. (2004). Science. 306(5694):279-283).
Species Reactivity	Human Mouse
Species Reactivity Note	Human, Mouse. Predicted to react with a broad range of species based on 100% sequence homology.
Antibody Type	Polyclonal Antibody
Entrez Gene Number	NP_003529
Gene Symbol	HIST1H4A H4/A ;H4FA HIST1H4B H4/I ;H4FI HIST1H4C H4/G H4FG HIST1H4D H4/B H4FB HIST1H4E H4/J H4FJ HIST1H4F H4/C H4FC HIST1H4H H4/H H4FH HIST1H4I H4/M H4FM HIST1H4J H4/E H4FE HIST1H4K H4/D H4FD HIST1H4L H4/K H4FK HIST2H4A H4/N H4F2 H4FN HIST2H4 HIST2H4B H4/O H4FO HIST4H4 HIST1H4A H4/A
Modifications	Citrullination
Purification Method	Affinity purified.
UniProt Number	P62805
Molecular Weight	~12 kDa observed. 11.24/11.37 kDa (H4 with/without Met1 removal) calculated. Uncharacterized bands may be observed in some lysate(s).

Biological Information

Immunogen

KLH-conjugated, synthetic peptide corresponding to amino acids 1-10 (SG[cit]GKGGKGL-C) of human histone H4 (Met1 removed), where [cit] denotes citrullinated Arg3. A C-terminal cysteine was added for conjugation purposes.

Epitope

N-terminus

Concentration

Please refer to lot specific datasheet.

Host

Rabbit

Specificity

This rabbit polyclonal antibody detected PAD4-catalyzed Arg3 citrullination of recombinant histone H4 and H2A, as well as calcium ionophore-induced histone H4 Arg3 citrullination (H4Cit3) in HL-60 cells. Cellular PAD4 (peptidyl arginine deiminase 4) knockdown greatly diminished ionophor-induced H4Cit3, while antibody blocking with the immunogen peptide prevented target band detection (Tanikawa, C., et al. (2012). Nat. Commun. 3:676; Wang, Y., et al. (2004). Science. 306(5694):279-283).

Species Reactivity

Human
Mouse

Species Reactivity Note

Human, Mouse. Predicted to react with a broad range of species based on 100% sequence homology.

Antibody Type

Polyclonal Antibody

Entrez Gene Number

NP_003529

Gene Symbol

HIST1H4A
H4/A
;H4FA
HIST1H4B
H4/I
;H4FI
HIST1H4C
H4/G
H4FG
HIST1H4D
H4/B
H4FB
HIST1H4E
H4/J
H4FJ
HIST1H4F
H4/C
H4FC
HIST1H4H
H4/H
H4FH
HIST1H4I
H4/M
H4FM
HIST1H4J
H4/E
H4FE
HIST1H4K
H4/D
H4FD
HIST1H4L
H4/K
H4FK
HIST2H4A
H4/N
H4F2
H4FN
HIST2H4
HIST2H4B
H4/O
H4FO
HIST4H4
HIST1H4A
H4/A

Modifications

Citrullination

Purification Method

Affinity purified.

UniProt Number

P62805

Molecular Weight

~12 kDa observed. 11.24/11.37 kDa (H4 with/without Met1 removal) calculated. Uncharacterized bands may be observed in some lysate(s).

Product Usage Statements
Quality Assurance	Evaluated by Western Blotting in HL-60 cell lysate. Western Blotting Analysis: A 1:1,000 dilution of this antibody detected differentiation-induced histone H4 Arg3 citrullination (H4Cit3) in 10 µg of lysate from calcium ionophore-treated HL-60 cells.
Usage Statement	Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

Product Usage Statements

Quality Assurance

Evaluated by Western Blotting in HL-60 cell lysate.

Western Blotting Analysis: A 1:1,000 dilution of this antibody detected differentiation-induced histone H4 Arg3 citrullination (H4Cit3) in 10 µg of lysate from calcium ionophore-treated HL-60 cells.

Usage Statement

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

Storage and Shipping Information
Storage Conditions	Stable for 1 year at 2-8°C from date of receipt.

Storage and Shipping Information

Storage Conditions

Stable for 1 year at 2-8°C from date of receipt.

Packaging Information
Material Size	200 µL

Packaging Information

Material Size

200 µL

实验耗材

分子生物学试剂

蛋白与免疫学

生化试剂

细胞及检测试剂

色谱及层析

仪器设备

实验服务

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Anti-Histone H4 (citrulline 3)

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