Anti-Phosphotyrosine Immuno affinity Purification Kit

Species Reactivity	Key Applications
H, M	WB, ChIP-seq, DB, ChIP

Species Reactivity

Key Applications

H, M

WB, ChIP-seq, DB, ChIP

Description
Catalogue Number	17-10493
Brand Family	Upstate
Trade Name	ChIPAb+ Upstate
Description	ChIPAb+ Trimethyl-Histone H3 (Lys36) - ChIP Validated Antibody and Primer Set
Overview	All ChIPAb+ antibodies are individually validated for chromatin precipitation, every lot, every time. Each ChIPAb+ antibody set includes control primers (tested every lot by qPCR) to biologically validate your IP results in a locus-specific context. The qPCR protocol and primer sequences are provided, allowing researchers to validate ChIP protocols when using our antibody in their chromatin context. Each set also includes a negative control antibody to ensure specificity of the ChIP reaction. The ChIPAb+ Trimethyl-Histone H3 (Lys36) set includes the Trimethyl-Histone H3 (Lys36) antibody, a negative control antibody (normal rabbit IgG), and qPCR primers which amplify a 147 bp region of human BDNF intron. The Trimethyl-Histone H3 (Lys36) and negative control antibodies are supplied in a scalable "per ChIP" reaction size and can be used to functionally validate the precipitation of Trimethyl-Histone H3 (Lys36)-associated chromatin.
Alternate Names	H3K36me2 Histone H3 (dimethyl K36) H3 histone family, member T Histone 3, H3 Histone cluster 3, H3
Background Information	Histones are highly conserved proteins that serve as the structural scaffold for the organization of nuclear DNA into chromatin. The four core histones, H2A, H2B, H3, and H4, assemble into an octamer (2 molecules of each). Subsequently, 146 base pairs of DNA are wrapped around the octamer, forming a nucleosome, the basic subunit of chromatin. Histones are modified post-translationally by the actions of enzymes in both the nucleus and cytoplasm. These modifications regulate DNA transcription, repair, recombination, and replication. The most commonly studied modifications are acetylation, phosphorylation, methylation, and ubiquitination. These modifications can alter local chromatin architecture, or recruit trans-acting factors that recognize specific histone modifications (the "histone code" hypothesis). The modifications occur predominantly on the N-terminal and C-terminal tails that extend beyond the nucleosome core particle. Methylation of histone H3 on Lys36 (H3K36me2/3) is tightly associated with actively transcribed genes, and this modification is found primarily within the coding region, suggesting H3K36 methylation is necessary for efficient RNA polymerase II elongation and processivity.

Description

Catalogue Number

17-10493

Brand Family

Upstate

Trade Name

ChIPAb+
Upstate

Description

ChIPAb+ Trimethyl-Histone H3 (Lys36) - ChIP Validated Antibody and Primer Set

Overview

All ChIPAb+ antibodies are individually validated for chromatin precipitation, every lot, every time. Each ChIPAb+ antibody set includes control primers (tested every lot by qPCR) to biologically validate your IP results in a locus-specific context. The qPCR protocol and primer sequences are provided, allowing researchers to validate ChIP protocols when using our antibody in their chromatin context. Each set also includes a negative control antibody to ensure specificity of the ChIP reaction.
The ChIPAb+ Trimethyl-Histone H3 (Lys36) set includes the Trimethyl-Histone H3 (Lys36) antibody, a negative control antibody (normal rabbit IgG), and qPCR primers which amplify a 147 bp region of human BDNF intron. The Trimethyl-Histone H3 (Lys36) and negative control antibodies are supplied in a scalable "per ChIP" reaction size and can be used to functionally validate the precipitation of Trimethyl-Histone H3 (Lys36)-associated chromatin.

Alternate Names

H3K36me2
Histone H3 (dimethyl K36)
H3 histone family, member T
Histone 3, H3
Histone cluster 3, H3

Background Information

Histones are highly conserved proteins that serve as the structural scaffold for the organization of nuclear DNA into chromatin. The four core histones, H2A, H2B, H3, and H4, assemble into an octamer (2 molecules of each). Subsequently, 146 base pairs of DNA are wrapped around the octamer, forming a nucleosome, the basic subunit of chromatin. Histones are modified post-translationally by the actions of enzymes in both the nucleus and cytoplasm. These modifications regulate DNA transcription, repair, recombination, and replication. The most commonly studied modifications are acetylation, phosphorylation, methylation, and ubiquitination. These modifications can alter local chromatin architecture, or recruit trans-acting factors that recognize specific histone modifications (the "histone code" hypothesis). The modifications occur predominantly on the N-terminal and C-terminal tails that extend beyond the nucleosome core particle. Methylation of histone H3 on Lys36 (H3K36me2/3) is tightly associated with actively transcribed genes, and this modification is found primarily within the coding region, suggesting H3K36 methylation is necessary for efficient RNA polymerase II elongation and processivity.

Product Information
Format	Purified
Control	Includes negative control rabbit IgG antibody and primers specific for human BDNF Intron.
Presentation	Anti-Trimethyl Histone H3 (Lys36) (Rabbit Polyclonal), Part No. CS207341. One vial containing 50 µL (0.05 mg/mL) purified rabbit polyclonal in buffer containing 0.1 M Tris-Glycine (pH7.4),150mM NaCl, and 0.05% sodium azide, before the addition of 30% glycerol. Store at -20°C. Normal Rabbit IgG, Part No. CS200581. One vial containing 7.5 µg Rabbit IgG in 75 µL storage buffer containing 0.05% sodium azide. Store at -20°C. ChIP Primers, GAPDH coding D2. Part No. CS207323. One vial containing 75 μL of 5 μM of each primer specific for human GAPDH coding region (chr12:6647453+6647539 hg19 build) . Store at -20°C. FOR: 5’ GCC ATG TAG ACC CCT TGA AGA G 3’ REV: 5’ ACT GGT TGA GCA CAG GGT ACT TTA T 3’

Product Information

Format

Purified

Control

Includes negative control rabbit IgG antibody and primers specific for human BDNF Intron.

Presentation

Anti-Trimethyl Histone H3 (Lys36) (Rabbit Polyclonal), Part No. CS207341. One vial containing 50 µL (0.05 mg/mL) purified rabbit polyclonal in buffer containing 0.1 M Tris-Glycine (pH7.4),150mM NaCl, and 0.05% sodium azide, before the addition of 30% glycerol. Store at -20°C.

Normal Rabbit IgG, Part No. CS200581. One vial containing 7.5 µg Rabbit IgG in 75 µL storage buffer containing 0.05% sodium azide. Store at -20°C.

ChIP Primers, GAPDH coding D2. Part No. CS207323. One vial containing 75 μL of 5 μM of each primer specific for human GAPDH coding region (chr12:6647453+6647539 hg19 build) . Store at -20°C.
FOR: 5’ GCC ATG TAG ACC CCT TGA AGA G 3’
REV: 5’ ACT GGT TGA GCA CAG GGT ACT TTA T 3’

Applications
Application	This ChIPAb+ Trimethyl-Histone H3 (Lys36) -ChIP Validated Antibody & Primer Set conveniently includes the antibody & the specific control PCR primers.
Key Applications	Western Blotting ChIP-seq Dot Blot Chromatin Immunoprecipitation (ChIP)
Application Notes	Chromatin Immunoprecipitation: Sonicated chromatin prepared from HeLa cells (1 X 10⁶ cell equivalents per IP) were subjected to chromatin immunoprecipitation using 2 µg of either Normal rabbit IgG or 2 µL Anti-trimethyl-Histone H3 (Lys36) and the Magna ChIP™ A Kit (Cat. # 17-610). Successful immunoprecipitation of trimethyl-Histone H3 (Lys36) associated DNA fragments was verified by qPCR using ChIP Primers, BDNF Intron as a positive locus, and GAPDH promoter primers as a negative locus (Please see figures). Data is presented as percent input of each IP sample relative to input chromatin for each amplicon and ChIP sample as indicated.Please refer to the EZ-Magna ChIP™ A (Cat. # 17-408) or EZ-ChIP™ (Cat. # 17-371) protocol for experimental details. Western Blot Analysis and Peptide Inhibition: Representative lot data. Recombinant Histone H3 (Catalog # 14-411, lane 1) and chicken Core Histones (Catalog # 13-107, lane 2) were resolved by electrophoresis, transferred to nitrocellulose and probed with antitrimethyl-Histone H3 (Lys36) (1:1000 dilution) or anti-trimethyl-Histone H3 (Lys36) pre-adsorbed with 1mM histone H3 peptides containing the following modifications: Lane 3: monomethyl-lysine 36 Lane 4: dimethyl-lysine 36 Lane 5: trimethyl-lysine 36 Lane 6: unmodified Proteins were visualized using a goat anti-rabbit secondary antibody conjugated to HRP and a chemiluminescence detection system. (Please see figures). Dot Blot Specificity Analysis: Representative lot data. Histone peptides with various modifications (see table) were transferred to PVDF membrane and probed with Anti-trimethyl Histone H3 (Lys36) (1:500 dilution). Proteins were visualized using a Donkey Anti-Rabbit IgG secondary antibody conjugated to HRP and a chemiluminescence detection system. (Please see figures).

Applications

Application

This ChIPAb+ Trimethyl-Histone H3 (Lys36) -ChIP Validated Antibody & Primer Set conveniently includes the antibody & the specific control PCR primers.

Key Applications

Western Blotting
ChIP-seq
Dot Blot
Chromatin Immunoprecipitation (ChIP)

Application Notes

Chromatin Immunoprecipitation:
Sonicated chromatin prepared from HeLa cells (1 X 10⁶ cell equivalents per IP) were subjected to chromatin immunoprecipitation using 2 µg of either Normal rabbit IgG or 2 µL Anti-trimethyl-Histone H3 (Lys36) and the Magna ChIP™ A Kit (Cat. # 17-610). Successful immunoprecipitation of trimethyl-Histone H3 (Lys36) associated DNA fragments was verified by qPCR using ChIP Primers, BDNF Intron as a positive locus, and GAPDH promoter primers as a negative locus (Please see figures). Data is presented as percent input of each IP sample relative to input chromatin for each amplicon and ChIP sample as indicated.Please refer to the EZ-Magna ChIP™ A (Cat. # 17-408) or EZ-ChIP™ (Cat. # 17-371) protocol for experimental details.

Western Blot Analysis and Peptide Inhibition:
Representative lot data.
Recombinant Histone H3 (Catalog # 14-411, lane 1) and chicken Core Histones (Catalog # 13-107, lane 2) were resolved by electrophoresis, transferred to nitrocellulose and probed with antitrimethyl-Histone H3 (Lys36) (1:1000 dilution) or anti-trimethyl-Histone H3 (Lys36) pre-adsorbed with 1mM histone H3 peptides containing the following modifications:
Lane 3: monomethyl-lysine 36
Lane 4: dimethyl-lysine 36
Lane 5: trimethyl-lysine 36
Lane 6: unmodified
Proteins were visualized using a goat anti-rabbit secondary antibody conjugated to HRP and a chemiluminescence detection system. (Please see figures).

Dot Blot Specificity Analysis:
Representative lot data.
Histone peptides with various modifications (see table) were transferred to PVDF membrane and probed with Anti-trimethyl Histone H3 (Lys36) (1:500 dilution). Proteins were visualized using a Donkey Anti-Rabbit IgG secondary antibody conjugated to HRP and a chemiluminescence detection system. (Please see figures).

Biological Information
Immunogen	Linear peptide corresponding to human Histone H3 trimethylated at Lys36 with peptide sequence APATGGV(K)KPHRYRPGC
Epitope	Trimethylated Lys36
Host	Rabbit
Specificity	Recognizes Trimethyl-Histone H3 (Lys36), Mr ~17 kDa.
Species Reactivity	Human Mouse
Species Reactivity Note	Wide reactivity with other species is expected.
Antibody Type	Polyclonal Antibody
Entrez Gene Number	NP_003484
Entrez Gene Summary	Histones are basic nuclear proteins that are responsiblefor the nucleosome structure of the chromosomal fiber in eukaryotes. Nucleosomes consist of approximately 146 bp of DNA wrapped around a histone octamer composed of pairs of each of the four core histones (H2A, H2B, H3, and H4). The chromatin fiber is further compacted through the interaction of a linker histone, H1,with the DNA between the nucleosomes to form higher order chromatin structures. This gene is intronless and encodes a member of the histone H3 family. Transcripts from this gene lack polyA tails; instead, they contain a palindromic termination element. This gene is located separately from the other H3 genes that are in the histone gene cluster on chromosome 6p22-p21.3. [provided by RefSeq]
Gene Symbol	H3.4 H3/g H3/t H3FT H3T H3t MGC126886 MGC126888 OTTHUMP00000037945
Modifications	Methylation
UniProt Number	Q16695
UniProt Summary	FUNCTION: Core component of nucleosome. Nucleosomes wrap and compact DNA into chromatin, limiting DNA accessibility to the cellular machineries which require DNA as a template. Histones thereby play a central role in transcription regulation, DNA repair, DNA replication and chromosomal stability. DNA accessibility is regulated via a complex set of post-translational modifications of histones, also called histone code, and nucleosome remodeling. SUBUNIT STRUCTURE: The nucleosome is a histone octamer containing two molecules each of H2A, H2B, H3 and H4 assembled in one H3-H4 heterotetramer and two H2A-H2B heterodimers. The octamer wraps approximately 147 bp of DNA. SUBCELLULAR LOCATION: Nucleus. TISSUE SPECIFICITY: Expressed in testicular cells. Developmental stage Expressed during S phase, then expression strongly decreases as cell division slows down during the process of differentiation. PTM: Acetylation is generally linked to gene activation. Acetylation on Lys-10 impairs methylation at Arg-9. Acetylation on Lys-19 and Lys-24 favors methylation at Arg-18 By similarity. Citrullination at Arg-9 and/or Arg-18 by PADI4 impairs methylation and represses transcription By similarity. Asymmetric dimethylation at Arg-18 by CARM1 is linked to gene activation. Symmetric dimethylation at Arg-9 by PRMT5 is linked to gene repression By similarity. Methylation at Lys-5, Lys-37 and Lys-80 are linked to gene activation. Methylation at Lys-5 facilitates subsequent acetylation of H3 and H4. Methylation at Lys-80 is associated with DNA double-strand break (DSB) responses and is a specific target for TP53BP1. Methylation at Lys-10 and Lys-28 are linked to gene repression. Methylation at Lys-10 is a specific target for HP1 proteins (CBX1, CBX3 and CBX5) and prevents subsequent phosphorylation at Ser-11 and acetylation of H3 and H4. Methylation at Lys-5 and Lys-80 require preliminary monoubiquitination of H2B at 'Lys-120'. Methylation at Lys-10 and Lys-28 are enriched in inactive X chromosome chromatin By similarity. Phosphorylated at Thr-4 by GSG2/haspin during prophase and dephosphorylated during anaphase. At centromeres, specifically phosphorylated at Thr-12 from prophase to early anaphase. Phosphorylated at Ser-11 during the whole mitosis. Phosphorylation at Ser-11, which is linked to gene activation, prevents methylation at Lys-10 but facilitates acetylation of H3 and H4. Phosphorylated at Ser-29 by MLTK isoform 1, RPS6KA5 or AURKB during mitosis or upon ultraviolet B irradiation By similarity. Phosphorylation at 'Ser-11' is crucial for chromosome condensation and cell-cycle progression during mitosis and meiosis. In addition phosphorylation at 'Ser-11' is important during interphase because it enables the transcription of genes following external stimulation, like stress or growth factors. Phosphorylation at 'Ser-11' is also an essential regulatory mechanism for neoplastic cell transformation. Phosphorylation at 'Ser-11' by AURKB/Aurora-B mediates the dissociation of HP1 proteins (CBX1, CBX3 and CBX5) from heterochromatin. Ubiquitinated By similarity. SIMILARITY:Belongs to the histone H3 family.
Molecular Weight	~17 kDa

Biological Information

Immunogen

Linear peptide corresponding to human
Histone H3 trimethylated at Lys36 with peptide sequence APATGGV(K)KPHRYRPGC

Epitope

Trimethylated Lys36

Host

Rabbit

Specificity

Recognizes Trimethyl-Histone H3 (Lys36), Mr ~17 kDa.

Species Reactivity

Human
Mouse

Species Reactivity Note

Wide reactivity with other species is expected.

Antibody Type

Polyclonal Antibody

Entrez Gene Number

NP_003484

Entrez Gene Summary

Histones are basic nuclear proteins that are responsiblefor the nucleosome structure of the chromosomal fiber in eukaryotes. Nucleosomes consist of approximately 146 bp of DNA wrapped around a histone octamer composed of pairs of each of the four core histones (H2A, H2B, H3, and H4). The chromatin fiber is further compacted through the interaction of a linker histone, H1,with the DNA between the nucleosomes to form higher order chromatin structures. This gene is intronless and encodes a member of the histone H3 family. Transcripts from this gene lack polyA tails; instead, they contain a palindromic termination element. This gene is located separately from the other H3 genes that are in the histone gene cluster on chromosome 6p22-p21.3. [provided by RefSeq]

Gene Symbol

H3.4
H3/g
H3/t
H3FT
H3T
H3t
MGC126886
MGC126888
OTTHUMP00000037945

Modifications

Methylation

UniProt Number

Q16695

UniProt Summary

FUNCTION: Core component of nucleosome. Nucleosomes wrap and compact DNA into chromatin, limiting DNA accessibility to the cellular machineries which require DNA as a template. Histones thereby play a central role in transcription regulation, DNA repair, DNA replication and chromosomal stability. DNA accessibility is regulated via a complex set of post-translational modifications of histones, also called histone code, and nucleosome remodeling.

SUBUNIT STRUCTURE: The nucleosome is a histone octamer containing two molecules each of H2A, H2B, H3 and H4 assembled in one H3-H4 heterotetramer and two H2A-H2B heterodimers. The octamer wraps approximately 147 bp of DNA.

SUBCELLULAR LOCATION: Nucleus.

TISSUE SPECIFICITY: Expressed in testicular cells.

Developmental stage Expressed during S phase, then expression strongly decreases as cell division slows down during the process of differentiation.

PTM: Acetylation is generally linked to gene activation. Acetylation on Lys-10 impairs methylation at Arg-9. Acetylation on Lys-19 and Lys-24 favors methylation at Arg-18 By similarity.

Citrullination at Arg-9 and/or Arg-18 by PADI4 impairs methylation and represses transcription By similarity.

Asymmetric dimethylation at Arg-18 by CARM1 is linked to gene activation. Symmetric dimethylation at Arg-9 by PRMT5 is linked to gene repression By similarity.

Methylation at Lys-5, Lys-37 and Lys-80 are linked to gene activation. Methylation at Lys-5 facilitates subsequent acetylation of H3 and H4. Methylation at Lys-80 is associated with DNA double-strand break (DSB) responses and is a specific target for TP53BP1. Methylation at Lys-10 and Lys-28 are linked to gene repression. Methylation at Lys-10 is a specific target for HP1 proteins (CBX1, CBX3 and CBX5) and prevents subsequent phosphorylation at Ser-11 and acetylation of H3 and H4. Methylation at Lys-5 and Lys-80 require preliminary monoubiquitination of H2B at 'Lys-120'. Methylation at Lys-10 and Lys-28 are enriched in inactive X chromosome chromatin By similarity.

Phosphorylated at Thr-4 by GSG2/haspin during prophase and dephosphorylated during anaphase. At centromeres, specifically phosphorylated at Thr-12 from prophase to early anaphase. Phosphorylated at Ser-11 during the whole mitosis. Phosphorylation at Ser-11, which is linked to gene activation, prevents methylation at Lys-10 but facilitates acetylation of H3 and H4. Phosphorylated at Ser-29 by MLTK isoform 1, RPS6KA5 or AURKB during mitosis or upon ultraviolet B irradiation By similarity.

Phosphorylation at 'Ser-11' is crucial for chromosome condensation and cell-cycle progression during mitosis and meiosis. In addition phosphorylation at 'Ser-11' is important during interphase because it enables the transcription of genes following external stimulation, like stress or growth factors. Phosphorylation at 'Ser-11' is also an essential regulatory mechanism for neoplastic cell transformation. Phosphorylation at 'Ser-11' by AURKB/Aurora-B mediates the dissociation of HP1 proteins (CBX1, CBX3 and CBX5) from heterochromatin.

Ubiquitinated By similarity.

SIMILARITY:Belongs to the histone H3 family.

Molecular Weight

~17 kDa

Product Usage Statements
Quality Assurance	Chromatin Immunoprecipitation: Sonicated chromatin prepared from HeLa cells (1e5 cell equivalents per IP) were subjected to chromatin immunoprecipitation using 0.1 µg of either Normal Rabbit IgG (Part No. CS200581), or 0.1 µg Anti-Trimethyl-Histone H3 (Lys36) (Part No.CS207341) and the Magna ChIP™ HiSens Kit (Cat. # 17-10460). Successful immunoprecipitation of trimethyl-Histone H3 (Lys36) associated DNA fragments was verified by qPCR using ChIP Primers, GAPDH coding D2 (Part No. CS207323). Please refer to the Magna ChIP™ HiSens (Cat. # 17-10460) or EZ-Magna ChIP™ HiSens (Cat. # 17-10461) protocol for experimental details.
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

Chromatin Immunoprecipitation:
Sonicated chromatin prepared from HeLa cells (1e5 cell equivalents per IP) were subjected to chromatin immunoprecipitation using 0.1 µg of either Normal Rabbit IgG (Part No. CS200581), or 0.1 µg Anti-Trimethyl-Histone H3 (Lys36) (Part No.CS207341) and the Magna ChIP™ HiSens Kit (Cat. # 17-10460). Successful immunoprecipitation of trimethyl-Histone H3 (Lys36) associated DNA fragments was verified by qPCR using ChIP Primers, GAPDH coding D2 (Part No. CS207323).
Please refer to the Magna ChIP™ HiSens (Cat. # 17-10460) or EZ-Magna ChIP™ HiSens (Cat. # 17-10461) protocol for experimental details.

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 -20°C from date of receipt. Handling Recommendations: Upon first thaw, and prior to removing the cap, centrifuge the vial and gently mix the solution. Aliquot into microcentrifuge tubes and store at -20°C. Avoid repeated freeze/thaw cycles, which may damage IgG and affect product performance.

Storage and Shipping Information

Storage Conditions

Stable for 1 year at -20°C from date of receipt. Handling Recommendations: Upon first thaw, and prior to removing the cap, centrifuge the vial and gently mix the solution. Aliquot into microcentrifuge tubes and store at -20°C. Avoid repeated freeze/thaw cycles, which may damage IgG and affect product performance.

Packaging Information
Material Size	25 assays
Material Package	25 assays per set. Recommended use: 0.1 μg of antibody per chromatin immunoprecipitation (dependent upon biological context).

Packaging Information

Material Size

25 assays

Material Package

25 assays per set. Recommended use: 0.1 μg of antibody per chromatin immunoprecipitation (dependent upon biological context).

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

办公用品

Anti-Phosphotyrosine Immuno affinity Purification Kit

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