Histone 3 anticorps (H3K36me3)

N° du produit ABIN2668403

Détail du produit anti-Histone 3 anticorps

Antigène: Histone 3 (H3)

Épitope: H3K36me3

Reactivité: Humain, Souris

Hôte: Souris

Clonalité: Monoclonal

Conjugué: Cet anticorp Histone 3 est non-conjugé

Application: 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)

Purification: Protein G Chromatography

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

Clone: MABI 0333

Isotype: IgG1

Information d'application

Indications d'application: 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.

Restrictions: For Research Use only

Independent Validation (CUT&RUN)

Validation #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

Date: 14.08.2023

Antigène: H3K36me3

Numéro du lot: 20822015

Application validée: Cleavage Under Targets and Release Using Nuclease

Contrôle positif:

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

Contrôle négative:

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

Conclusion:

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.

Protocole

Anticorps primaire: ABIN2668403

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.

Stockage

Concentration: 0.44 μg/μL

Buffer: PBS pH 7.5 containing 30 % glycerol, 0.3 M NaCl, and 0.035 % sodium azide.

Agent conservateur: Sodium azide

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

Conseil sur la manipulation:

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

Stock: -20 °C

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

Date de péremption: 6 months

Références pour anticorps anti-Histone 3 (ABIN2668403)

Brahma, Henikoff: "RSC-Associated Subnucleosomes Define MNase-Sensitive Promoters in Yeast." dans: 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." dans: 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." dans: 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." dans: PLoS genetics, Vol. 6, Issue 9, pp. e1001091, (2010) (PubMed).

Détails sur Histone 3

Antigène: Histone 3 (H3)

Autre désignation: Histone H3 (H3 Produits)

Synonymes: anticorps H-3, anticorps histocompatibility 3, anticorps H3

Poids moléculaire: 17 kDa

ID gène: 3020