HDAC1 Antikörper

Produktnummer ABIN2854776

Produktdetails anti-HDAC1 Antikörper

Target: HDAC1 (Histone Deacetylase 1 (HDAC1))

Reaktivität: Human

Wirt: Kaninchen

Klonalität: Polyklonal

Konjugat: Dieser HDAC1 Antikörper ist unkonjugiert

Applikation: 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))

Kreuzreaktivität: Human, Maus, Ratte, Zebrafisch (Danio rerio)

Produktmerkmale: Rabbit Polyclonal antibody to HDAC1 (histone deacetylase 1)
HDAC1 antibody

Aufreinigung: Purified by antigen-affinity chromatography.

Güteklasse: KO Validated

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

Isotyp: IgG

Anwendungsinformationen

Applikationshinweise: 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.

Kommentare:

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

Validation: KO/KD, Orthogonal, Overexpression

Beschränkungen: Nur für Forschungszwecke einsetzbar

Independent Validation (CUT&RUN)

Validierung #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

Datum: 28.02.2022

Antigen: HDAC1

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

Validierungsbilder

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.

Protokoll

Primärantikörper: 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.

Anmerkungen:

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

Handhabung

Format: Liquid

Konzentration: 1 mg/mL

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

Konservierungsmittel: Thimerosal (Merthiolate)

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

Lagerung: 4 °C,-20 °C

Informationen zur Lagerung: 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.

Referenzen für anti-HDAC1 Antikörper (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." in: 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." in: 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." in: 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." in: 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." in: 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." in: 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." in: Frontiers in pharmacology, Vol. 7, pp. 81, (2016) (PubMed).

Details zu HDAC1

Target: HDAC1 (Histone Deacetylase 1 (HDAC1))

Andere Bezeichnung: histone deacetylase 1 (HDAC1 Produkte)

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

Hintergrund: 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

Molekulargewicht: 55 kDa

Gen-ID: 3065

UniProt: Q13547

Pathways: Neurotrophin Signalübertragung, 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