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Histone 3 抗体 (H3K36me3)

Name: Histone 3 抗体 (H3K36me3)
SKU: ABIN2668403
Availability: OutOfStock
Rating: 5 (1 reviews)

This anti-Histone 3 antibody is a 小鼠单克隆 antibody detecting Histone 3 in WB, ChIP, DB, ChIP-seq, CUT&RUN 和 CUT&Tag. Suitable for 人和小鼠. Independently validated for use in Cleavage Under Targets and Release Using Nuclease. This Primary Antibody has been cited in 4+ publications.

Cleavage Under Targets and Release Using Nuclease validation image for anti-Histone 3 (H3) (H3K36me3) antibody (ABIN2668403) (Histone 3 抗体 (H3K36me3))

CUT&RUN

Western blot of Histone H3 trimethyl Lys36 antibody. (Histone 3 抗体 (H3K36me3))

Dot blot of Histone H3 trimethyl Lys36 antibody. (Histone 3 抗体 (H3K36me3))

ChIP-seq

Histone H3 dimethyl Lys36 antibody (pAb) tested by dot blot analysis. (Histone 3 抗体 (H3K36me3))

Histone H3 dimethyl Lys36 antibody tested by Western blot. (Histone 3 抗体 (H3K36me3))

7 images

(4 references)

(1 validation)

产品编号 ABIN2668403

发货至: 中国

Datasheet as PDF

Quick Overview for Histone 3 抗体 (H3K36me3) (ABIN2668403)

抗原

See all Histone 3 (H3) 抗体

Histone 3 (H3)

适用

1344
908
811
41
39
39
34
33
26
23
21
21
6
5
4
4
3
3
3
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

小鼠

克隆类型

单克隆

标记

This Histone 3 antibody is un-conjugated

应用范围

1028
418
374
323
252
201
188
187
163
159
148
126
115
58
48
48
35
22
15
13
7
7
6
4
3
1
1
1
1

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

克隆位点

MABI 0333

Discover our top product H3 Primary Antibody

抗原表位
52

50

48

44

39

37

37

36

35

35

34

33

32

31

31

30

23

23

22

22

22

19

18

17

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16

16

16

16

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15

14

13

13

13

13

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12

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10

9

9

9

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8

8
H3K36me3

纯化方法

Protein G Chromatography

免疫原

This Histone H3 trimethylLys36 antibody was raised against a peptide containing trimethylLys36 of human Histone H3.

亚型

IgG1
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, DB, ICC, 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, DB, ICC, ChIP-seq, CUT&RUN 宿主: 兔 Polyclonal unconjugated

anti-Histone 3 (H3) (H3K9ac) antibody
H3 适用: 人, 小鼠 WB, IF, ChIP, DB, ICC, ChIP-seq, CUT&RUN, CUT&Tag 宿主: 兔 Polyclonal unconjugated

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

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

anti-Histone 3 (H3) (H3K9me2) antibody
H3 适用: 人 WB, IF, IHC, ChIP, IP, ChIP-seq 宿主: 兔 Polyclonal unconjugated

anti-Histone 3 (H3) (H3K9me3) antibody
H3 适用: 人 WB, IF, IHC, ChIP, DB 宿主: 兔 Polyclonal unconjugated

应用备注

Recommended starting concentrations are
ChIP: 5 - 10 µg per ChIP
ChIP-Seq: 5 - 10 µg each
WB: 0.5 - 2 µg/mL dilution
DB: 0.5 - 2 µg/mL dilution
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 #104510 (Cleavage Under Targets and Release Using Nuclease)

by

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

No.

#104510

日期

2023.08.14

抗原

H3K36me3

Lot Number

20822015

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. ABIN2668403 allows for specific targeting of H3K36me3 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-H3K36me3 antibody ABIN2668403 (left) after library preparation, compared to the E-Gel Sizing DNA Ladder (Thermo Fisher) (right).

1. Alignment tracks from CUT&RUN targeting H3K36me3 in human fibroblast cells using antibody ABIN2668403 showing the USP32 locus. 2. Alignment tracks for CUT&RUN with the IgG negative control ABIN101961. 3. RefSeq Genes.
Full Methods
Primary Antibody

ABIN2668403

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-H3K36me3 antibody ABIN2668403, 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

0.44 μg/μL

缓冲液

PBS pH 7.5 containing 30 % glycerol, 0.3 M NaCl, 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
Brahma, Henikoff: "RSC-Associated Subnucleosomes Define MNase-Sensitive Promoters in Yeast." in: Molecular cell, Vol. 73, Issue 2, pp. 238-249.e3, (2019) (PubMed).

Powers, Parvanov, Baker, Walker, Petkov, Paigen: "The Meiotic Recombination Activator PRDM9 Trimethylates Both H3K36 and H3K4 at Recombination Hotspots In Vivo." in: PLoS genetics, Vol. 12, Issue 6, pp. e1006146, (2016) (PubMed).

Xu, Gan, Zhou, Wee, Zhang, Ito: "Arabidopsis MRG domain proteins bridge two histone modifications to elevate expression of flowering genes." in: Nucleic acids research, Vol. 42, Issue 17, pp. 10960-74, (2014) (PubMed).

Rechtsteiner, Ercan, Takasaki, Phippen, Egelhofer, Wang, Kimura, Lieb, Strome: "The histone H3K36 methyltransferase MES-4 acts epigenetically to transmit the memory of germline gene expression to progeny." in: PLoS genetics, Vol. 6, Issue 9, pp. e1001091, (2010) (PubMed).
抗原

Histone 3 (H3)

别名

Histone H3

分子量

17 kDa

基因ID

3020