Histone 3 Antikörper (H3K27ac)

Produktnummer ABIN2668475

Produktdetails anti-Histone 3 Antikörper

Target: Histone 3 (H3)

Bindungsspezifität: H3K27ac

Reaktivität: Human, Saccharomyces cerevisiae

Wirt: Kaninchen

Klonalität: Polyklonal

Konjugat: Dieser Histone 3 Antikörper ist unkonjugiert

Applikation: 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)

Aufreinigung: Protein A Chromatography

Immunogen: This Histone H3 acetylLys27 antibody was raised against a peptide including acetyllysine 27 of histone H3.

Isotyp: IgG

Anwendungsinformationen

Applikationshinweise: Recommended starting concentrations are
ChIP: 10 µg per ChIP
ChIP-Seq: 5 µg each
ICC/IF: 1 - 5 µg/mL dilution
WB: 0.1 - 1 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.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Independent Validation (CUT&RUN)

Validierung #104489 (Cleavage Under Targets and Release Using Nuclease)

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

No.: #104489

Datum: 14.08.2023

Antigen: H3K27ac

Chargennummer: 31521015

Validierte Anwendung: Cleavage Under Targets and Release Using Nuclease

Positivkontrolle:

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

Negativkontrolle:

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

Bewertung:

Passed. ABIN2668475 allows for specific targeting of H3K27ac in human cells using CUT&RUN.

Validierungsbilder

Library profiles comparing fragment size distributions on an E-Gel EX 2% agarose gel (Thermo Fisher). Fragments obtained from CUT&RUN using anti-H3K27ac antibody ABIN2668475 (left) after library preparation, compared to the E-Gel Sizing DNA Ladder (Thermo Fisher) (right).

1. Alignment tracks from CUT&RUN targeting H3K27ac in human fibroblast cells using antibody ABIN2668475 showing the SNF8 locus. 2. Alignment tracks for CUT&RUN with the IgG negative control ABIN101961. 3. RefSeq Genes.

Protokoll

Primärantikörper: ABIN2668475

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.
• Cell 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-H3K27ac antibody ABIN2668475, 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.

Handhabung

Konzentration: 1 μg/μL

Konservierungsmittel: Sodium azide

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

Handhabung:

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

Lagerung: -20 °C

Informationen zur Lagerung: Antibodies in solution can be stored at -20 °C for 2 years.

Haltbarkeit: 6 months

Referenzen für anti-Histone 3 Antikörper (ABIN2668475)

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Details zu Histone 3

Target: Histone 3 (H3)

Andere Bezeichnung: Histone H3 (H3 Produkte)

Synonyme: H-3 antikoerper, histocompatibility 3 antikoerper, H3 antikoerper

Hintergrund: Histone H3 is one of the core components of the nucleosome. The nucleosome is the smallest subunit of chromatin and consists of 147 base pairs of DNA wrapped around an octamer of core histone proteins (two each of Histone H2A, Histone H2B, Histone H3 and Histone H4). Chromatin is subject to a variety of chemical modifications, including post- translational modifications of the histone proteins and the methylation of cytosine residues in the DNA. Reported histone modifications include acetylation, methylation, phosphorylation, ubiquitylation, glycosylation, ADP-ribosylation, carbonylation and SUMOylation, these modifications play a major role in regulating gene expression. Lysine 27 of histone H3 can also be mono-, di- or trimethylated by different histone methyltransferases, such as EZH2 or NSD3. While histone methylation can be associated with transcriptional activation or repression, methylation of Lysine 27 of histone H3 is mainly associated with transcriptional repression.

Molekulargewicht: 17 kDa

Gen-ID: 3020