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Histone 3 抗体 (H3K9ac)

Name: Histone 3 抗体 (H3K9ac)
SKU: ABIN2668415
Availability: OutOfStock
Rating: 5 (1 reviews)

H3 适用: 人, 小鼠 WB, IF, ChIP, ICC, DB, ChIP-seq, CUT&RUN, CUT&Tag 宿主: 兔 Polyclonal unconjugated

Cleavage Under Targets and Release Using Nuclease validation image for anti-Histone 3 (H3) (H3K9ac) antibody (ABIN2667854) (Histone 3 抗体 (H3K9ac))

CUT&RUN

Western blot of Histone H3 acetyl Lys9 antibody (pAb). (Histone 3 抗体 (H3K9ac))

ChIP

Histone H3 acetyl Lys9 antibody tested by dot blot analysis. (Histone 3 抗体 (H3K9ac))

ChIP-seq

Immunofluorescence stain of Histone H3 acetyl Lys9 pAb. (Histone 3 抗体 (H3K9ac))

7 images

(3 references)

(1 validation)

产品编号 ABIN2668415

发货至: 中国

Datasheet as PDF

抗原 See all Histone 3 (H3) 抗体

Histone 3 (H3)
抗原表位
57

49

47

42

40

39

36

36

35

34

34

33

32

30

28

27

26

23

23

23

22

22

20

18

18

17

16

16

16

15

15

15

15

14

13

13

13

12

12

12

11

11

10

10

10

10

9

9

8

8
H3K9ac
适用
1386

956

877

59

42

41

41

40

39

35

35

30

26

22

20

6

3

3

3

3

2

2

2

2

2

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1
人, 小鼠
宿主
1203

240

8

5
兔
Compatible Secondaries
克隆类型
1127

328

1
多克隆
标记
797

80

48

48

48

48

48

48

34

33

21

18

18

18

17

17

17

17

17

17

17

17

12

1
This Histone 3 antibody is un-conjugated
应用范围
1035

408

374

315

241

188

173

166

163

153

138

127

117

48

38

34

32

22

15

13

7

7

6

4

3

1

1

1
Western Blotting (WB), Immunofluorescence (IF), Chromatin Immunoprecipitation (ChIP), Immunocytochemistry (ICC), Dot Blot (DB), ChIP DNA-Sequencing (ChIP-seq), Cleavage Under Targets and Release Using Nuclease (CUT&RUN), Cleavage Under Targets and Tagmentation (CUT&Tag)

纯化方法

Protein A Chromatography

免疫原

This Histone H3 acetylLys9 antibody was raised against a peptide including acetyllysine 9 of histone H3.

亚型

IgG

Top Product

Discover our top product H3 Primary Antibody
anti-Histone 3 (H3) (H3K4me) antibody
H3 适用: 人 WB, IF, ChIP, IP, ChIP-seq, CUT&RUN 宿主: 兔 Polyclonal unconjugated

anti-Histone 3 (H3) (H3K27ac) antibody
H3 适用: 人, Saccharomyces cerevisiae WB, IF, ChIP, ICC, DB, ChIP-seq, CUT&RUN, CUT&Tag 宿主: 兔 Polyclonal unconjugated

anti-Histone 3 (H3) (3meLys4) antibody
H3 适用: 人, Saccharomyces cerevisiae WB, IF, ChIP, ICC, DB, ChIP-seq, CUT&RUN 宿主: 兔 Polyclonal unconjugated

anti-Histone 3 (H3) (H3K36me3) antibody
H3 适用: 人, 小鼠 WB, ChIP, DB, ChIP-seq, CUT&RUN, CUT&Tag 宿主: 小鼠 Monoclonal MABI 0333 unconjugated

anti-Histone 3 (H3) (AA 71-136) antibody
H3 适用: 人, 小鼠, 大鼠 WB, ELISA, ICC, FACS, IHC (p), IF (cc), IF (p), IHC (fro) 宿主: 兔 Polyclonal unconjugated

anti-Histone 3 (H3) (H3K27me) antibody
H3 适用: 人 WB, IHC, IF, ChIP, IP, ChIP-seq 宿主: 兔 Polyclonal unconjugated

anti-Histone 3 (H3) (H3K4me3) antibody
H3 适用: 人, 小鼠, Saccharomyces cerevisiae WB, IF, ChIP, ICC, DB, ChIP-seq, CUT&RUN, CUT&Tag 宿主: 兔 Polyclonal unconjugated

anti-Histone 3 (H3) (C-Term) antibody
H3 适用: 人 WB, IHC, ChIP, IP 宿主: 兔 Polyclonal unconjugated

anti-Histone 3 (H3) (H3K4me3) antibody
H3 适用: 人 WB, IHC, IF, ChIP, IP, DB, ChIP-seq, CUT&RUN, CUT&Tag 宿主: 兔 Polyclonal unconjugated

anti-Histone 3 (H3) (H3K79me) antibody
H3 适用: 人 WB, IHC, IF, ChIP, IP, ChIP-seq 宿主: 兔 Polyclonal unconjugated

应用备注

Recommended starting concentrations are
ChIP: 10 µg per ChIP
ChIP-Seq: 3 µg each
ICC/IF: 2 µg/mL dilution
WB: 0.5 - 2 µg/mL dilution
CUT&RUN: 1:100
CUT&Tag: 1 µg/50 µL reaction
CUT&RUN: 2 µL/200 µL reaction
Optimal working dilution should be determined by the investigator.

限制

仅限研究用
生效 #104509 (Cleavage Under Targets and Release Using Nuclease)

by

Anna Nordin and Claudio Cantù; Cantù Lab, Gene Regulation during Development and Disease, Linköping University

No.

#104509

日期

2023.08.14

抗原

H3K9ac

Lot Number

165220066

Method validated

Cleavage Under Targets and Release Using Nuclease

Positive Control

Polyclonal rabbit anti-H3K4me (antibodies-online, ABIN3023251)

Negative Control

Polyclonal guinea pig anti-rabbit IgG (antibodies-online, ABIN101961)

Notes

Passed. ABIN2667854 allows for specific targeting of H3K9ac in human cells using CUT&RUN.

Validation Images

Library profiles comparing fragment size distributions on an E-Gel EX 2% agarose gel (Thermo Fisher). Fragments obtained from CUT&RUN using anti-H3K9ac antibody ABIN2667854 (left) after library preparation, compared to the E-Gel Sizing DNA Ladder (Thermo Fisher) (right).

1. Alignment tracks from CUT&RUN targeting H3K9ac in human fibroblast cells using antibody ABIN2667854 showing the VMP1 locus. 2. Alignment tracks for CUT&RUN with the IgG negative control ABIN101961. 3. RefSeq Genes.
Full Methods
Primary Antibody

ABIN2667854

Secondary Antibody

Full Protocol

Cell harvest
Harvest 50,000 human fibroblast cells per antibody.
Centrifuge cell solution 3 min at 600 x g at RT.
Remove the liquid carefully.
Gently resuspend cells in 1 mL of Nuclear Extraction Buffer (20 mM HEPES-KOH pH 8.2, 20% Glycerol, 0,05% IGEPAL, 0.5 mM Spermidine, 10 mM KCl, Roche Complete Protease Inhibitor EDTA-free).
Move the solution to a 2 mL centrifuge tube.
Pellet the nuclei 800 x g for 5 min.
Repeat the NE Buffer wash twice for a total of three washes.
Resuspend the nuclei in 20 µL NE Buffer per sample.
Concanavalin A beads preparation
Prepare one 2 mL microcentrifuge tube.
Gently resuspend the magnetic Concanavalin A Beads (antibodies-online, ABIN6923139).
Pipette 10 µL Con A Beads slurry for each sample into the 1.5 mL microcentrifuge tube.
Place the tube on a magnet stand until the fluid is clear. Remove the liquid carefully.
Remove the microcentrifuge tube from the magnetic stand.
Pipette 1 mL Binding Buffer (20 mM HEPES pH 7.5, 10 mM KCl, 1 mM CaCl₂, 1 mM MnCl₂) into each tube and resuspend ConA beads by gentle pipetting.
Spin down the liquid from the lid with a quick pulse in a table-top centrifuge.
Place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
Remove the microcentrifuge tube from the magnetic stand.
Repeat twice for a total of three washes.
Gently resuspend the ConA Beads in a volume of Binding Buffer corresponding to the original volume of bead slurry, i.e. 10 µL per sample.
Nuclei immobilization – binding to Concanavalin A beads
Carefully vortex the nuclei suspension and add 10 µL of the Con A beads in Binding Buffer to the cell suspension for each sample.
Close tube tightly incubates 10 min at 4 °C.
Put the 2 mL tube on the magnet stand and when the liquid is clear remove the supernatant.
Resuspend the beads in 1 mL of EDTA wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, Roche Complete Protease Inhibitor EDTA-free, 2mM EDTA).
Incubate 5 min at RT.
Place the tube on the magnet stand and when the liquid is clear remove the supernatant.
Resuspend the beads in 200µl of Wash Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, Roche Complete Protease Inhibitor EDTA-free) for each sample.
Cell permeabilization and primary antibody binding
Divide nuclei suspension into separate PCR tubes, one for each antibody (200 µL per sample).
Add 2 µL antibody (anti-H3K9ac antibody ABIN2667854, anti-H3K4me positive control antibody ABIN3023251, and guinea pig anti-rabbit IgG negative control antibody ABIN101961) to the respective tube, corresponding to a 1:100 dilution.
Incubate ON at 4 °C.
Place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
Remove the microcentrifuge tubes from the magnetic stand.
Wash with 200 µL of Wash Buffer using a multichannel pipette to accelerate the process.
Repeat the wash five times for a total of six washes.
pAG-MNase Binding
Prepare a 1.5 mL microcentrifuge tube containing 200 µL of pAG mix for each sample (200 µl of wash buffer + 120 ng pAG-MNase per sample).
Place the PCR tubes with the sample on a magnet stand until the fluid is clear. Remove the liquid carefully.
Remove tubes from the magnetic stand.
Resuspend the beads in 200 µL of pAG-MNase premix.
Incubate for 30 min at 4 °C.
Place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
Remove the microcentrifuge tubes from the magnetic stand.
Wash with 200 µL of Wash Buffer using a multichannel pipette to accelerate the process.
Repeat the wash for a total of five washes.
Resuspend in 200 µL of Wash Buffer.
MNase digestion and release of pAG-MNase-antibody-chromatin complexes
Place PCR tubes on ice and allow to chill.
Prepare a 1.5 mL microcentrifuge tube with 51 µl of 2 mM CaCl₂ mix per sample (50 µl Wash Buffer + 1 µL 100 mM CaCl₂) and let it chill on ice.
Always in ice, place the samples on the magnetic rack and when the liquid is clear remove the supernatant.
Resuspend the samples in 50 µl of the 2 mM CaCl₂ mix and incubate in ice for exactly 30 min.
Place the sample on the magnet stand and when the liquid is clear move the supernatant in fresh collection tubes with 3µl of EDTA/EGTA 0.25M (Digestion buffer).
Resuspend the sample in 47 µl of 1x Urea STOP Buffer (8.5 M Urea, 100 mM NaCl, 2 mM EGTA, 2 mM EDTA, 0,5% IGEPAL).
Incubate the samples 1 h at 4 °C.
Transfer the supernatant containing the pAG-MNase-bound digested chromatin fragments to the previously collected digestion buffer.
DNA Clean up
Take the Mag-Bind® TotalPure NGS beads (Omega Bio-Tek, M1378-01) from the storage and wait until they are at RT.
Add 2x volume of beads to each sample (e.g. 100 µL of beads for 50 µL of sample).
Incubate the beads and the sample for 15 min at RT.
During incubation prepare fresh EtOH 80%.
Place the PCR tubes on a magnet stand and when the liquid is clear remove the supernatant.
Add 200 µl of fresh 80% EtOH to the sample without disturbing the beads (Important!!! Do NOT resuspend the beads or remove the tubes from the magnet stand or the sample will be lost).
Incubate 30 sec at RT.
Remove the EtOH from the sample.
Repeat the wash with 80% EtOH.
Resuspend the beads in 25 µL of 10 mM Tris.
Incubate the sample for 2 min at RT.
Repeat the 2x beads clean up as described before (this time with 50 µL of beads for each sample).
Resuspend the beads + DNA in 20 µL of 10 mM Tris.
Library preparation and sequencing
Prepare libraries using KAPA HyperPrep Kit using KAPA Dual-Indexed adapters according to protocol.
Sequence samples on an Illumina NextSeq 500 sequencer, using a NextSeq 500/550 High Output Kit v2.5 (75 Cycles), 36bp PE.
Bioinformatics
Align reads the human genome (hg38) using bowtie78 with settings -X 700 -m1 -v 3. Remove duplicate reads, and sort files using samtools. Filter mapped reads for size, keeping only reads with a fragment size at or below 120 base pairs.
Generate bedgraph files using bedtools genomecov.
Call peaks using SEACR version 1.3, in relaxed mode, normalizing to the negative control.

Experimental Notes
浓度

1 μg/μL

缓冲液

Purified IgG in PBS ( pH 7.5) with 30 % glycerol and 0.035 % sodium azide.

储存液

Sodium azide

注意事项

This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.

注意事项

Avoid repeated freeze/thaw cycles and keep on ice when not in storage.

储存条件

-20 °C

储存方法

Antibodies in solution can be stored at -20 °C for 2 years.

有效期

6 months
Zhang, Li, Rezaeian, Xu, Chou, Jin, Han, Pan, Wang, Long, Zhang, Huang, Tsai, Tsai, Logothetis, Lin: "H3 ubiquitination by NEDD4 regulates H3 acetylation and tumorigenesis." in: Nature communications, Vol. 8, pp. 14799, (2017) (PubMed).

Holmberg Olausson, Nistér, Lindström: "Loss of nucleolar histone chaperone NPM1 triggers rearrangement of heterochromatin and synergizes with a deficiency in DNA methyltransferase DNMT3A to drive ribosomal DNA transcription." in: The Journal of biological chemistry, Vol. 289, Issue 50, pp. 34601-19, (2014) (PubMed).

Begum, Stevens, Smith, Connor, Challis, Bloomfield, White: "Epigenetic changes in fetal hypothalamic energy regulating pathways are associated with maternal undernutrition and twinning." in: FASEB journal : official publication of the Federation of American Societies for Experimental Biology, Vol. 26, Issue 4, pp. 1694-703, (2012) (PubMed).
抗原

Histone 3 (H3)

别名

Histone H3 (H3 产品)

别名

H-3 antibody, histocompatibility 3 antibody, H3 antibody

分子量

17 kDa

基因ID

3020