S6 KIN ASSAY KIT 200 ASSAYS

Species Reactivity	Key Applications
H, Vrt	WB, IP, EMSA, ChIP

Species Reactivity

Key Applications

H, Vrt

WB, IP, EMSA, ChIP

Description
Catalogue Number	17-10136
Trade Name	ChIPAb+ Upstate
Description	ChIPAb+ Dimethyl-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+™ Dimethyl Histone H3 (Lys36) set includes a Dimethyl Histone H3 (Lys36) antibody, a Normal Rabbit Serum, and positive control primers which amplify a 87 bp region of the human GAPDH coding region. The Dimethyl Histone H3 (Lys36) antibody and negative controls are supplied in a scalable "per ChIP" reaction size and can be used to functionally validate the precipitation of dimethyl 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

17-10136

Trade Name

ChIPAb+
Upstate

Description

ChIPAb+ Dimethyl-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+™ Dimethyl Histone H3 (Lys36) set includes a Dimethyl Histone H3 (Lys36) antibody, a Normal Rabbit Serum, and positive control primers which amplify a 87 bp region of the human GAPDH coding region. The Dimethyl Histone H3 (Lys36) antibody and negative controls are supplied in a scalable "per ChIP" reaction size and can be used to functionally validate the precipitation of dimethyl 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	Serum
Control	Includes normal rabbit serum and primers specific for human GAPDH, coding region.
Presentation	Anti-Dimethyl Histone H3 (Lys36) (Rabbit Polyclonal), Part No. CS207357. One vial containing 25 µL rabbit polyclonal serum containing 0.05% sodium azide, before the addition of 30% glycerol. Store at -20°C. Normal Rabbit Serum, Part No. CS200585. One vial containing 25 µL antiserum 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, hg 19 build) Store at -20°C. FOR: 5’ GCC ATG TAG ACC CCT TGA AGA GC 3’ REV: 5’ ACT GGT TGA GCA CAG GGT ACT TTA T 3’

Product Information

Format

Serum

Control

Includes normal rabbit serum and primers specific for human GAPDH, coding region.

Presentation

Anti-Dimethyl Histone H3 (Lys36) (Rabbit Polyclonal), Part No. CS207357. One vial containing 25 µL rabbit polyclonal serum containing 0.05% sodium azide, before the addition of 30% glycerol. Store at -20°C.

Normal Rabbit Serum, Part No. CS200585. One vial containing 25 µL antiserum 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, hg 19 build) Store at -20°C.
FOR: 5’ GCC ATG TAG ACC CCT TGA AGA GC 3’
REV: 5’ ACT GGT TGA GCA CAG GGT ACT TTA T 3’

Applications
Application	This ChIPAb+ Validated Antibody & Primer Set conveniently includes the antibody, matched IgG negative control antibody & set control PCR primers that detect a known positive locus.
Key Applications	Western Blotting Immunoprecipitation Electrophoretic Mobility Shift Assay Chromatin Immunoprecipitation (ChIP)
Application Notes	Chromatin Immunoprecipitation: Sonicated chromatin prepared from HeLa cells (1e5 cell equivalents per IP) were subjected to chromatin immunoprecipitation using 1 µL of either Normal Rabbit Serum (Part No. CS200585), or 1 µL Anti-Dimethyl Histone H3 (Lys36) (Part No.CS207357) and the Magna ChIP™ HiSens (Cat. # 17-10460). Successful immunoprecipitation of dimethyl Histone H3 (Lys36) associated DNA fragments was verified by qPCR using ChIP Primers, GAPDH coding D2 (Part No. CS207323) as a positive locus, and GAPDH promoter (Part No. 22-004) as a negative locus. (Figure 2). Data are presented as percent input of each IP sample relative to input chromatin for each amplicon and ChIP sample as indicated. Please refer to the Magna ChIP™ HiSens (Cat. # 17-10460) or EZ-MagnaChIP™ HiSens(Cat. # 17-10461) protocol for experimental details. Chromatin Immunoprecipitation: Sonicated chromatin prepared from HeLa cells (1e5 cell equivalents per IP) were subjected to chromatin immunoprecipitation using 1 µL of either Normal Rabbit Serum (Part No. CS200585), or 1 µL Anti-Dimethyl Histone H3 (Lys36) (Part No.CS207357) and the Magna ChIP™ HiSens (Cat. # 17-10460). Successful immunoprecipitation of dimethyl Histone H3 (Lys36) associated DNA fragments was verified by qPCR using ChIP Primers, GAPDH coding D2 (Part No. CS207323) as a positive locus, and GAPDH promoter (Part No. 22-004) as a negative locus. Please refer to the Magna ChIP™ HiSens (Cat. # 17-10460) or EZ-MagnaChIP™ HiSens(Cat. # 17-10461) protocol for experimental details. Western Blotting Analysis: A 1:10,000 dilution of this antibody detected dimethyl Histone H3 (Lys36) in 10 µg HeLa acid extract and demonstrated a loss of signal in 0.5 µg of unmethylated recombinant Histone H3 Dot Blot Analysis: Representative lot data. Absurance™ Histone H3 Antibody Specificity Array (Cat. No. 16-667, Fig. A) Absurance™ Histone H2A, H2B, and H4 Antibody Specificity Array (Cat. No. 16-665, Fig. B) were probed with Anti-Dimethyl Histone H3 (Lys36) (1:10,000 dilution). Proteins were visualized using a Donkey Anti-Rabbit IgG secondary antibody conjugated to HRP and a chemiluminescence detection system.

Applications

Application

This ChIPAb+ Validated Antibody & Primer Set conveniently includes the antibody, matched IgG negative control antibody & set control PCR primers that detect a known positive locus.

Key Applications

Western Blotting
Immunoprecipitation
Electrophoretic Mobility Shift Assay
Chromatin Immunoprecipitation (ChIP)

Application Notes

Chromatin Immunoprecipitation:
Sonicated chromatin prepared from HeLa cells (1e5 cell equivalents per IP) were subjected to chromatin immunoprecipitation using 1 µL of either Normal Rabbit Serum (Part No. CS200585), or 1 µL Anti-Dimethyl Histone H3 (Lys36) (Part No.CS207357) and the Magna ChIP™ HiSens (Cat. # 17-10460). Successful immunoprecipitation of dimethyl Histone H3 (Lys36) associated DNA fragments was verified by qPCR using ChIP Primers, GAPDH coding D2 (Part No. CS207323) as a positive locus, and GAPDH promoter (Part No. 22-004) as a negative locus. (Figure 2). Data are presented as percent input of each IP sample relative to input chromatin for each amplicon and ChIP sample as indicated.
Please refer to the Magna ChIP™ HiSens (Cat. # 17-10460) or EZ-MagnaChIP™ HiSens(Cat. # 17-10461) protocol for experimental details.

Chromatin Immunoprecipitation:
Sonicated chromatin prepared from HeLa cells (1e5 cell equivalents per IP) were subjected to chromatin immunoprecipitation using 1 µL of either Normal Rabbit Serum (Part No. CS200585), or 1 µL Anti-Dimethyl Histone H3 (Lys36) (Part No.CS207357) and the Magna ChIP™ HiSens (Cat. # 17-10460). Successful immunoprecipitation of dimethyl Histone H3 (Lys36) associated DNA fragments was verified by qPCR using ChIP Primers, GAPDH coding D2 (Part No. CS207323) as a positive locus, and GAPDH promoter (Part No. 22-004) as a negative locus. Please refer to the Magna ChIP™ HiSens (Cat. # 17-10460) or EZ-MagnaChIP™ HiSens(Cat. # 17-10461) protocol for experimental details.

Western Blotting Analysis:
A 1:10,000 dilution of this antibody detected dimethyl Histone H3 (Lys36) in 10 µg HeLa acid extract and demonstrated a loss of signal in 0.5 µg of unmethylated recombinant Histone H3

Dot Blot Analysis: Representative lot data.
Absurance™ Histone H3 Antibody Specificity Array (Cat. No. 16-667, Fig. A) Absurance™ Histone H2A, H2B, and H4 Antibody Specificity Array (Cat. No. 16-665, Fig. B) were probed with Anti-Dimethyl Histone H3 (Lys36) (1:10,000 dilution). Proteins were visualized using a Donkey Anti-Rabbit IgG secondary antibody conjugated to HRP and a chemiluminescence detection system.

Biological Information
Immunogen	KLH-conjugated linear peptide corresponding to a region near the N-terminus of human Histone H3 dimethylated at Lys36.
Epitope	Dimethylated Lys36
Host	Rabbit
Specificity	Recognizes Dimethyl Histone H3 (Lys36). No detectable cross-reactivity unmethylated, mono, or dimethylated Lys36. No detectable cross reactivity to other histone modifications as measured by AbSurance Histone Antibody Specificity Arrays.
Species Reactivity	Human Vertebrates
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

KLH-conjugated linear peptide corresponding to a region near the N-terminus of human Histone H3 dimethylated at Lys36.

Epitope

Dimethylated Lys36

Host

Rabbit

Specificity

Recognizes Dimethyl Histone H3 (Lys36). No detectable cross-reactivity unmethylated, mono, or dimethylated Lys36. No detectable cross reactivity to other histone modifications as measured by AbSurance Histone Antibody Specificity Arrays.

Species Reactivity

Human
Vertebrates

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	Sonicated chromatin prepared from HeLa cells (1e5 cell equivalents per IP) were subjected to chromatin immunoprecipitation using 1 µL of either Normal Rabbit Serum (Part No. CS200585), or 1 µL Anti-Dimethyl Histone H3 (Lys36) (Part No.CS207357) and the Magna ChIP™ HiSens Kit (Cat. # 17-10460). Successful immunoprecipitation of dimethyl-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

Sonicated chromatin prepared from HeLa cells (1e5 cell equivalents per IP) were subjected to chromatin immunoprecipitation using 1 µL of either Normal Rabbit Serum (Part No. CS200585), or 1 µL Anti-Dimethyl Histone H3 (Lys36) (Part No.CS207357) and the Magna ChIP™ HiSens Kit (Cat. # 17-10460). Successful immunoprecipitation of dimethyl-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	Recommended use: 1 μL of antibody per chromatin immunoprecipitation (dependent upon biological context).

Packaging Information

Material Size

25 assays

Material Package

Recommended use: 1 μL 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

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