HDAC1 anticorps

N° du produit ABIN2854776

Détail du produit anti-HDAC1 anticorps

Antigène: HDAC1 (Histone Deacetylase 1 (HDAC1))

Reactivité: Humain

Hôte: Lapin

Clonalité: Polyclonal

Conjugué: Cet anticorp HDAC1 est non-conjugé

Application: Western Blotting (WB), Immunofluorescence (IF), Immunohistochemistry (Paraffin-embedded Sections) (IHC (p)), Immunoprecipitation (IP), Immunocytochemistry (ICC), Chromatin Immunoprecipitation (ChIP), Immunohistochemistry (Frozen Sections) (IHC (fro)), Immunohistochemistry (Whole Mount) (IHC (wm))

Réactivité croisée: Humain, Souris, Rat, Poisson zèbre (Danio rerio)

Attributs du produit: Rabbit Polyclonal antibody to HDAC1 (histone deacetylase 1)
HDAC1 antibody

Purification: Purified by antigen-affinity chromatography.

Classe de qualité: KO Validated

Immunogène: Recombinant protein encompassing a sequence within the center region of human HDAC1. The exact sequence is proprietary.

Isotype: IgG

Information d'application

Indications d'application: WB: 1:500-1:3000. ICC/IF: 1:100-1:1000. IHC-P: 1:100-1:1000. IP: 1:100-1:500. Optimal dilutions/concentrations should be determined by the researcher. Not tested in other applications.

Commentaires:

Positive Control: 293T , A431 , HeLa , HepG2 , U87-MG , SK-N-SH , Rat-2 , NIH3T3 , DDDDK-tagged HDAC1-transfected 293T

Validation: KO/KD, Orthogonal, Overexpression

Restrictions: For Research Use only

Independent Validation (CUT&RUN)

Validation #104404 (Cleavage Under Targets and Release Using Nuclease)

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

No.: #104404

Date: 28.02.2022

Antigène: HDAC1

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. ABIN2854776 allows for HDAC1 targeted digestion using CUT&RUN in mouse fore limbs (11.5) cells.

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-HDAC1 antibody ABIN2854776 after library preparation, compared to the E-Gel Sizing DNA Ladder (Thermo Fisher).

1. Alignment tracks from CUT&RUN targeting HDAC1 in Mouse fore limb (11.5) cells using anti-HDAC1 antibody ABIN2854776. 2. Peaks called by SEACR from CUT&RUN data using anti HDAC1 ABIN2854776. 3. RefSeq Genes.

Protocole

Anticorps primaire: ABIN2854776

Full Protocol:

• Cell harvest and nuclear extraction
• • Dissect 3 Fore limbs (11.5 DAC) from mouse strain RjOrl:SWISS for each sample.
  • Dissociate the tissue into single cells in TrypLE for 15 min at 37 °C.
  • Centrifuge cell solution 5 min at 800 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 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, ABIN6952467).
  • Pipette 20 µL Con A Beads slurry for each sample into the 2 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 the 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 the wash 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. 20 µL per sample.
• Nuclei immobilization – binding to Concanavalin A beads
• • Carefully vortex the nuclei suspension and add 20 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 200 µl of Wash Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, Roche Complete Protease Inhibitor EDTA-free) per sample.
• Primary antibody binding
• • Divide nuclei suspension into separate 200 µL PCR tubes, one for each antibody.
  • Add 2 µL antibody (anti-HDAC1 antibody ABIN2854776, anti-H3K27me3 antibody positive control ABIN6923144, and guinea pig anti-rabbit IgG negative control antibody ABIN101961) to the respective tube, corresponding to a 1:100 dilution.
  • Incubate at 4 °C ON.
  • 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 100 µL of pAG mix per sample (100 µL of wash buffer + 58.5 µg 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 100 µL of pAG-MNase premix.
  • Incubate 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 five times for a total of six washes.
  • Resuspend in 100 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 102 µl of 2 mM CaCl₂ mix per sample (100 µl Wash Buffer + 2 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 100 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 remove the supernatant.
  • Resuspend the sample in 50 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 1h at 4°C.
  • Transfer the supernatant containing the pAG-MNase-bound digested chromatin fragments to fresh 200 µl PCR tubes.
• 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), 36 bp PE.
• Peak calling
• • Trim reads using using bbTools bbduk (BBMap - Bushnell B. - sourceforge.net/projects/bbmap/) to remove adapters, artifacts and repeat sequences.
  • Map aligned reads to the hg38 human genome using bowtie with options -m 1 -v 0 -I 0 -X 500.
  • Use SAMtools to convert SAM files to BAM files and remove duplicates.
  • Use BEDtools genomecov to produce Bedgraph files.
  • Call peaks using SEACR with a 0.001 threshold and the option norm stringent.

Notes:

The protocol is published in Zambanini, G. et al. A New CUT&RUN Low Volume-Urea (LoV-U) protocol uncovers Wnt/β-catenin tissue-specific genomic targets. bioRxiv (2022). https://doi.org/10.1101/2022.07.06.498999

Stockage

Format: Liquid

Concentration: 1 mg/mL

Buffer: 1XPBS ( pH 7), 20 % Glycerol, 0.01 % Thimerosal

Agent conservateur: Thimerosal (Merthiolate)

Précaution d'utilisation: This product contains Thimerosal (Merthiolate): a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.

Stock: 4 °C,-20 °C

Stockage commentaire: Store as concentrated solution. Centrifuge briefly prior to opening vial. For short-term storage (1-2 weeks), store at 4°C. For long-term storage, aliquot and store at -20°C or below. Avoid multiple freeze-thaw cycles.

Références pour anticorps anti-HDAC1 (ABIN2854776)

Zambanini, Nordin, Jonasson, Pagella, Cantù: "A new cut&run low volume-urea (LoV-U) protocol optimized for transcriptional co-factors uncovers Wnt/b-catenin tissue-specific genomic targets." dans: Development (Cambridge, England), (2022) (PubMed).

Hu, Chung, Ping, Hsu, Tsai, Chen, Cheng: "Differential Expression of Multiple Disease-Related Protein Groups Induced by Valproic Acid in Human SH-SY5Y Neuroblastoma Cells." dans: Brain sciences, Vol. 10, Issue 8, (2020) (PubMed).

Ochiai, Hayashi, Umeda, Yoshimura, Harada, Shimizu, Nakano, Saitoh, Liu, Yamamoto, Okamura, Ohkawa, Kimura, Nikaido: "Genome-wide kinetic properties of transcriptional bursting in mouse embryonic stem cells." dans: Science advances, Vol. 6, Issue 25, pp. eaaz6699, (2020) (PubMed).

Lin, Wang, Wu, Lin, Chen, Chen, Chen, Peng: "Nifedipine Exacerbates Lipogenesis in the Kidney via KIM-1, CD36, and SREBP Upregulation: Implications from an Animal Model for Human Study." dans: International journal of molecular sciences, Vol. 21, Issue 12, (2020) (PubMed).

Deng, Yang, Ji, Lu, Qiu, Sheng, Sun, Kong: "Overexpression of peptidase inhibitor 16 attenuates angiotensin II-induced cardiac fibrosis via regulating HDAC1 of cardiac fibroblasts." dans: Journal of cellular and molecular medicine, Vol. 24, Issue 9, pp. 5249-5259, (2020) (PubMed).

Lee, Lin, Chae, Yoo, Kim, Lee, Johnson, Kim, Cantley, Lee, Yu, Cho: "The chromatin remodeler RSF1 controls centromeric histone modifications to coordinate chromosome segregation." dans: Nature communications, Vol. 9, Issue 1, pp. 3848, (2019) (PubMed).

Lee, Kuo, Tsai, Cheng, Chen, Chan, Lin, Lin, Li, Kanwar, Leung, Cheung, Huang, Wang, Cheung: "Inhibition of HDAC3- and HDAC6-Promoted Survivin Expression Plays an Important Role in SAHA-Induced Autophagy and Viability Reduction in Breast Cancer Cells." dans: Frontiers in pharmacology, Vol. 7, pp. 81, (2016) (PubMed).

Détails sur HDAC1

Antigène: HDAC1 (Histone Deacetylase 1 (HDAC1))

Autre désignation: histone deacetylase 1 (HDAC1 Produits)

Synonymes: anticorps GON-10, anticorps HD1, anticorps RPD3, anticorps RPD3L1, anticorps CG7471, anticorps DHDAC1, anticorps DRpd3, anticorps DmHDAC1, anticorps Dmel\\CG7471, anticorps E(var)3-64BC, anticorps HDAC, anticorps HDAC-1, anticorps HDAC1, anticorps Hdac1, anticorps Rpd3/HDAC, anticorps Su(var)3-26, anticorps Su(var)326, anticorps Su(var)328, anticorps dHDAC-1, anticorps dHDAC1, anticorps dRPD3, anticorps dRpd3, anticorps dmHDA401, anticorps drpd3, anticorps hdac1, anticorps l(3)04556, anticorps l(3)64Cc, anticorps rpd3, anticorps rpd[3], anticorps gon-10, anticorps hdac1b, anticorps rpd3l1, anticorps Rpd3, anticorps GB14706, anticorps hdac1-b, anticorps chunp6919, anticorps hdac-1, anticorps mp:zf637-2-001987, anticorps wu:fb19h11, anticorps wu:fi06f03, anticorps zgc:101582, anticorps zgc:65818, anticorps Hdac1-ps, anticorps MommeD5, anticorps ARABIDOPSIS HISTONE DEACETYLASE 1, anticorps ARABIDOPSIS HISTONE DEACETYLASE 19, anticorps ATHD1, anticorps ATHDA19, anticorps ATRPD3A, anticorps F20D10.250, anticorps F20D10_250, anticorps HDA1, anticorps HDA19, anticorps HISTONE DEACETYLASE, anticorps HISTONE DEACETYLASE 19, anticorps HISTONE DEACETYLASE19, anticorps RPD3A, anticorps histone deacetylase 1, anticorps HDM, anticorps ab21, anticorps hdac1a, anticorps histone deacetylase 1, anticorps Histone deacetylase 1, anticorps histone deacetylase, anticorps histone deacetylase 1 S homeolog, anticorps histone deacetylase Rpd3, anticorps histone deacetylase 1 L homeolog, anticorps HDAC1, anticorps hdac1.S, anticorps LOC411503, anticorps hdac1, anticorps Hdac1, anticorps HD1, anticorps hda-1, anticorps hdac1.L, anticorps LOC748850

Sujet: Histone acetylation and deacetylation, catalyzed by multisubunit complexes, play a key role in the regulation of eukaryotic gene expression. The protein encoded by this gene belongs to the histone deacetylase/acuc/apha family and is a component of the histone deacetylase complex. It also interacts with retinoblastoma tumor-suppressor protein and this complex is a key element in the control of cell proliferation and differentiation. Together with metastasis-associated protein-2, it deacetylates p53 and modulates its effect on cell growth and apoptosis.

Cellular Localization: Nucleus

Poids moléculaire: 55 kDa

ID gène: 3065

UniProt: Q13547

Pathways: Neurotrophin Signaling Pathway, Intracellular Steroid Hormone Receptor Signaling Pathway, Regulation of Intracellular Steroid Hormone Receptor Signaling, Mitotic G1-G1/S Phases, Regulation of Muscle Cell Differentiation, Skeletal Muscle Fiber Development, Negative Regulation of intrinsic apoptotic Signaling, Embryonic Body Morphogenesis