TUNEL Apoptosis Detection Kit (DNA Fragmentation/Fluorescence Staining)

Species Reactivity	Key Applications
B, H, M, Yeast (S. cerevisiae)	WB, ChIP

Species Reactivity

Key Applications

B, H, M, Yeast (S. cerevisiae)

WB, ChIP

Description
Catalogue Number	17-10241
Trade Name	ChIPAb+ Upstate
Description	ChIPAb+™ Acetyl-Histone H3 (Lys9/18) - 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+ Acetyl-Histone H3 (Lys9/18) set includes the Acetyl-Histone H3 (Lys9/18) antibody, a Normal rabbit serum, and control primers which amplify a 166 bp region of human GAPDH promoter. The Acetyl-Histone H3 (Lys9/18) and negative controls are supplied in a scalable "per ChIP" reaction size and can be used to functionally validate the precipitation of Acetyl-Histone H3 (Lys9/18)-associated chromatin.
Alternate Names	H3K9/18Ac Histone H3 (acetyl K9/18)
Background Information	Histone H3 is one of the 5 main histone proteins involved in the structure of chromatin in eukaryotic cells. Featuring a main globular domain and a long N terminal tail H3 is involved with the structure of the nucleosomes of the 'beads on a string' structure. Acetylation of histone H3 occurs at several different lysine positions in the histone tail and is performed by a family of enzymes known as Histone Acetyl Transferases (HATs).

Description

Catalogue Number

17-10241

Trade Name

ChIPAb+
Upstate

Description

ChIPAb+™ Acetyl-Histone H3 (Lys9/18) - 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+ Acetyl-Histone H3 (Lys9/18) set includes the Acetyl-Histone H3 (Lys9/18) antibody, a Normal rabbit serum, and control primers which amplify a 166 bp region of human GAPDH promoter. The Acetyl-Histone H3 (Lys9/18) and negative controls are supplied in a scalable "per ChIP" reaction size and can be used to functionally validate the precipitation of Acetyl-Histone H3 (Lys9/18)-associated chromatin.

Alternate Names

H3K9/18Ac
Histone H3 (acetyl K9/18)

Background Information

Histone H3 is one of the 5 main histone proteins involved in the structure of chromatin in eukaryotic cells. Featuring a main globular domain and a long N terminal tail H3 is involved with the structure of the nucleosomes of the 'beads on a string' structure.

Acetylation of histone H3 occurs at several different lysine positions in the histone tail and is performed by a family of enzymes known as Histone Acetyl Transferases (HATs).

Product Information
Format	Serum
Control	Includes normal rabbit serum and primers specific for human GAPDH promoter.
Presentation	Anti-Acetyl-Histone H3 (Lys9/18) (rabbit polyclonal), . One vial containing 25 µL of rabbit antiserum containing 0.05% sodium azide before the addition of glycerol to 30%. Store at -20°C. Normal Rabbit Serum, . One vial containing 25 μL of antiserum containing 0.05% sodium azide. Store at -20°C. Control Primers, human GAPDH promoter, . One vial containing 75 μL of 5 μM of each primer specific for human GAPDH. Store at -20°C. FOR: TAC TAG CGG TTT TAC GGG CG REV: TCG AAC AGG AGG AGC AGA GAG CGA

Product Information

Format

Serum

Control

Includes normal rabbit serum and primers specific for human GAPDH promoter.

Presentation

Anti-Acetyl-Histone H3 (Lys9/18) (rabbit polyclonal), . One vial containing 25 µL of rabbit antiserum containing 0.05% sodium azide before the addition of glycerol to 30%. Store at -20°C.
Normal Rabbit Serum, . One vial containing 25 μL of antiserum containing 0.05% sodium azide. Store at -20°C.
Control Primers, human GAPDH promoter, . One vial containing 75 μL of 5 μM of each primer specific for human GAPDH. Store at -20°C.
FOR: TAC TAG CGG TTT TAC GGG CG
REV: TCG AAC AGG AGG AGC AGA GAG CGA

Applications
Application	This ChIPAb+ Acetyl-Histone H3 (Lys9/18) -ChIP Validated Antibody & Primer Set conveniently includes the antibody & the specific control PCR primers.
Key Applications	Western Blotting Chromatin Immunoprecipitation (ChIP)
Application Notes	Chromatin Immunoprecipitation: Representative lot data. Sonicated chromatin prepared from HeLa cells (1 X 10E6 cell equivalents per IP) were subjected to chromatin immunoprecipitation using 1 µL of either Normal Rabbit Serum, or 1 µL of Anti-Acetyl-Histone H3 (Lys9/18), and the Magna ChIP™ A Kit (Cat. # 17-610). Successful immunoprecipitation of acetyl-Histone H3 (Lys9/18) associated DNA fragments was verified by qPCR using Control Primers specific for the human GAPDH promoter region as a positive locus and human β-globin primers as a negative locus. (Figure 2). 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: Representative lot data. Acid extracts from untreated (lane 1), sodium butyrate treated (lane 2, Catalog # 17-305) HeLa cells and recombinant Histone H3, (Lane 3, Catalog # 14-411) were resolved by electrophoresis, transferred to nitrocellulose and probed with Anti-Acetyl-Histone H3 (Lys9/18) (1:5,000 dilution). Proteins were visualized using a goat anti-rabbit secondary antibody conjugated to HRP and a chemiluminescence detection system. Arrow indicates Acetyl-histone H3 (Lys9/18) (~17kDa). (Figure 3). Beadlyte® Histone-Peptide Specificity Assay: Representative lot data. 1:1,000-1:40,000 dilutions of anti-acetyl-Histone H3 (Lys9/18) were incubated with a cocktail of microspheres conjugated to histone H3 peptides with the following modifications: 1. acetyl-lysine 4 2. acetyl-lysine 14 3. acetyl-lysine 9 4. acetyl-lysine 18 5. No modifications, containing aa 1-20 Unbound antibody was removed by filtration. The peptide-antibody complexes were incubated with a biotin-conjugated anti-mouse secondary antibody followed by incubation with a phycoerythrin-streptavidin conjugate. Fluorescence was read on a Luminex® 100™ instrument. Median fluorescence intensity (MFI) is plotted. (Figure 4).

Applications

Application

This ChIPAb+ Acetyl-Histone H3 (Lys9/18) -ChIP Validated Antibody & Primer Set conveniently includes the antibody & the specific control PCR primers.

Key Applications

Western Blotting
Chromatin Immunoprecipitation (ChIP)

Application Notes

Chromatin Immunoprecipitation:
Representative lot data.
Sonicated chromatin prepared from HeLa cells (1 X 10E6 cell equivalents per IP) were subjected to chromatin immunoprecipitation using 1 µL of either Normal Rabbit Serum, or 1 µL of Anti-Acetyl-Histone H3 (Lys9/18), and the Magna ChIP™ A Kit (Cat. # 17-610). Successful immunoprecipitation of acetyl-Histone H3 (Lys9/18) associated DNA fragments was verified by qPCR using Control Primers specific for the human GAPDH promoter region as a positive locus and human β-globin primers as a negative locus. (Figure 2). 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:
Representative lot data.
Acid extracts from untreated (lane 1), sodium butyrate treated (lane 2, Catalog # 17-305) HeLa cells and recombinant Histone
H3, (Lane 3, Catalog # 14-411) were resolved by electrophoresis, transferred to nitrocellulose and probed with Anti-Acetyl-Histone H3 (Lys9/18) (1:5,000 dilution). Proteins were visualized using a goat anti-rabbit secondary antibody conjugated to HRP and a chemiluminescence detection system.
Arrow indicates Acetyl-histone H3 (Lys9/18) (~17kDa). (Figure 3).
Beadlyte® Histone-Peptide Specificity Assay:
Representative lot data.
1:1,000-1:40,000 dilutions of anti-acetyl-Histone H3 (Lys9/18) were incubated with a cocktail of microspheres conjugated to histone H3 peptides with the following modifications:
1. acetyl-lysine 4
2. acetyl-lysine 14
3. acetyl-lysine 9
4. acetyl-lysine 18
5. No modifications, containing aa 1-20
Unbound antibody was removed by filtration. The peptide-antibody complexes were incubated with a biotin-conjugated anti-mouse secondary antibody followed by incubation with a phycoerythrin-streptavidin conjugate.
Fluorescence was read on a Luminex® 100™ instrument. Median
fluorescence intensity (MFI) is plotted. (Figure 4).

Biological Information
Immunogen	KLH-conjugated synthetic peptide (..ARAcKSTGGKAPRAcKQL..) in which AcK corresponds to acetyl-lysine at residue 9 and 18 of human Histone H3.
Epitope	Acetylated Lys9/18
Host	Rabbit
Specificity	Recognizes Histone H3 acetylated on lysines 9 and 18.
Species Reactivity	Bovine Human Mouse Yeast (S. cerevisiae)
Species Reactivity Note	Broad species cross-reactivity expected
Antibody Type	Polyclonal Antibody
Entrez Gene Number	NM_003493
Entrez Gene Summary	Histones are basic nuclear proteins that are responsible for the nucleosome structure of the chromosomal fiber in eukaryotes. Two molecules of each of the four core histones (H2A, H2B, H3, and H4) form an octamer, around which approximately 146 bp of DNA is wrapped in repeating units, called nucleosomes. The linker histone, H1, interacts with linker DNA between nucleosomes and functions in the compaction of chromatin into higher order structures. This gene contains introns and its mRNA is polyadenylated, unlike most histone genes. The protein encoded is a replication-independent member of the histone H3 family.
Gene Symbol	H3F3A MGC87783 H3.3A MGC87782 H3F3 H3.3B H3F3B
Modifications	Acetylation
Purification Method	Unpurified
UniProt Number	Q16695
UniProt Summary	FUNCTION: SwissProt: Q16695 # 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. SIZE: 136 amino acids; 15508 Da SUBUNIT: 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. 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: SwissProt: Q16695 ## Belongs to the histone H3 family.
Molecular Weight	17 kDa

Biological Information

Immunogen

KLH-conjugated synthetic peptide (..ARAcKSTGGKAPRAcKQL..) in which AcK corresponds to acetyl-lysine at residue 9 and 18 of human Histone H3.

Epitope

Acetylated Lys9/18

Host

Rabbit

Specificity

Recognizes Histone H3 acetylated on lysines 9 and 18.

Species Reactivity

Bovine
Human
Mouse
Yeast (S. cerevisiae)

Species Reactivity Note

Broad species cross-reactivity expected

Antibody Type

Polyclonal Antibody

Entrez Gene Number

NM_003493

Entrez Gene Summary

Histones are basic nuclear proteins that are responsible for the nucleosome structure of the chromosomal fiber in eukaryotes. Two molecules of each of the four core histones (H2A, H2B, H3, and H4) form an octamer, around which approximately 146 bp of DNA is wrapped in repeating units, called nucleosomes. The linker histone, H1, interacts with linker DNA between nucleosomes and functions in the compaction of chromatin into higher order structures. This gene contains introns and its mRNA is polyadenylated, unlike most histone genes. The protein encoded is a replication-independent member of the histone H3 family.

Gene Symbol

H3F3A
MGC87783
H3.3A
MGC87782
H3F3
H3.3B
H3F3B

Modifications

Acetylation

Purification Method

Unpurified

UniProt Number

Q16695

UniProt Summary

FUNCTION: SwissProt: Q16695 # 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.
SIZE: 136 amino acids; 15508 Da
SUBUNIT: 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.
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: SwissProt: Q16695 ## Belongs to the histone H3 family.

Molecular Weight

17 kDa

Product Usage Statements
Quality Assurance	Chromatin Immunoprecipitation: Representative lot data. Sonicated chromatin prepared from HeLa cells (1 X 10E6 cell equivalents per IP) were subjected to chromatin immunoprecipitation using 1 µL of either Normal Rabbit Serum,or 1 µL of Anti-Acetyl-Histone H3 (Lys9/18) and the Magna ChIP™ A Kit (Cat. # 17-610). Successful immunoprecipitation of Acetyl-Histone H3 (Lys9/18) associated DNA fragments was verified by qPCR using Control Primers specific for the human GAPDH promoter region as a positive locus (Figure 1). Please refer to the EZ-Magna ChIP™ A (Cat. # 17-408) or EZ-ChIP™ (Cat. # 17-371) 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

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. Note: Variability in freezer temperatures below -20°C may cause glycerol containing solutions to become frozen during storage.

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.
Note: Variability in freezer temperatures below -20°C may cause glycerol containing solutions to become frozen during storage.

Packaging Information
Material Size	25 assays
Material Package	25 assays per set. Recommended use: ~1 μL of antibody per chromatin immunoprecipitation (dependent upon biological context).

Packaging Information

Material Size

25 assays

Material Package

25 assays per set. 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

办公用品

TUNEL Apoptosis Detection Kit (DNA Fragmentation/Fluorescence Staining)

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