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NIPBL anticorps (AA 2651-2805)

NIPBL Reactivité: Rat IF (cc), IF (p), IHC (p), IHC (fro), ICC Hôte: Lapin Polyclonal unconjugated
N° du produit ABIN1714202
  • Antigène Voir toutes NIPBL Anticorps
    NIPBL (Nipped-B like Protein (NIPBL))
    Épitope
    • 14
    • 5
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    AA 2651-2805
    Reactivité
    • 15
    • 15
    • 5
    Rat
    Hôte
    • 24
    • 3
    • 2
    • 1
    Lapin
    Clonalité
    • 26
    • 4
    Polyclonal
    Conjugué
    • 12
    • 3
    • 2
    • 2
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    Cet anticorp NIPBL est non-conjugé
    Application
    • 12
    • 12
    • 9
    • 8
    • 5
    • 4
    • 4
    • 4
    • 2
    • 1
    Immunofluorescence (Cultured Cells) (IF (cc)), Immunofluorescence (Paraffin-embedded Sections) (IF (p)), Immunohistochemistry (Paraffin-embedded Sections) (IHC (p)), Immunohistochemistry (Frozen Sections) (IHC (fro)), Immunocytochemistry (ICC)
     Réactivité croisée
    Rat
    Homologie
    Human,Mouse,Dog,Cow,Sheep,Pig,Horse,Chicken
    Purification
    Purified by Protein A.
    Immunogène
    KLH conjugated synthetic peptide derived from human IDN3
    Isotype
    IgG
  • Indications d'application
    IHC-P 1:200-400
    IHC-F 1:100-500
    IF(IHC-P) 1:50-200
    IF(IHC-F) 1:50-200
    IF(ICC) 1:50-200
    ICC 1:100-500
    CUT&RUN 1:100
    Restrictions
    For Research Use only
  • Validation #104411 (Cleavage Under Targets and Release Using Nuclease)
    'Independent Validation' signe
    by
    Gianluca Zambanini, Anna Nordin and Claudio Cantù; Cantù Lab, Gene Regulation during Development and Disease, Linköping University
    No.
    #104411
    Date
    26.04.2023
    Antigène
    NIPBL
    Numéro du lot
    AG10167864
    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. The anti-NIPBL ABIN1714202 allows for CUT&RUN targeted profiling of NIPBL binding in mouse forelimb cells.

    'Independent Validation' signe
    Validation Images
    Protocole
    Anticorps primaire
    ABIN1714202
    Anticorps secondaire
    Full Protocol
    • Cell harvest and nuclear extraction
      • Dissect 3 Fore limbs (11.5 DAC) from RjOrl:SWISS embryos 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 1.5 mL tube on the magnet rack 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, 2 mM EDTA).
      • Incubate for 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 (150,000 cells per sample).
      • Add 2 µL antibody (anti-NIPBL antibody ABIN1714202, anti-H3K4me positive control antibody ABIN3023251, 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 (to accelerate the process use a multichannel pipette).
      • Repeat the wash for a total of five washes.
    • pAG-MNase Binding
      • Prepare a 1.5 mL microcentrifuge tube containing 200 µL of pAG mix pear 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 3 µL of EDTA/EGTA 0.25 M (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 for 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 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.
      • 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 and 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 mm10 mouse 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. Development (2022). PMID 36355069

  • Format
    Liquid
    Concentration
    1 μg/μL
    Buffer
    0.01M TBS( pH 7.4) with 1 % BSA, 0.02 % Proclin300 and 50 % Glycerol.
    Agent conservateur
    ProClin
    Précaution d'utilisation
    This product contains ProClin: a POISONOUS AND HAZARDOUS SUBSTANCE, which should be handled by trained staff only.
    Stock
    4 °C,-20 °C
    Stockage commentaire
    Shipped at 4°C. Store at -20°C for one year. Avoid repeated freeze/thaw cycles.
    Date de péremption
    12 months
  • Antigène
    NIPBL (Nipped-B like Protein (NIPBL))
    Autre désignation
    IDN3 (NIPBL Produits)
    Synonymes
    anticorps CDLS, anticorps CDLS1, anticorps IDN3, anticorps IDN3-B, anticorps Scc2, anticorps SCC2W, anticorps scc2, anticorps nipbl, anticorps scc2-2, anticorps NIPBL, anticorps nipbla, anticorps wu:fi33d08, anticorps 4921518A06Rik, anticorps 4933421G18Rik, anticorps C79399, anticorps Idn3, anticorps NIPBL, cohesin loading factor, anticorps Nipped-B homolog-like, anticorps NIPBL, cohesin loading factor S homeolog, anticorps nipped-B homolog b (Drosophila), anticorps nipped-B-like protein A, anticorps Nipped-B homolog (Drosophila), anticorps NIPBL, anticorps NIPBLL, anticorps nipbl.S, anticorps LOC427439, anticorps nipblb, anticorps LOC100164947, anticorps nipbl, anticorps Nipbl
    Sujet

    Synonyms: CDLS, Colon tumor susceptibility 2, Delangin, DKFZp434L1319, FLJ11203, FLJ12597, FLJ13354, FLJ13648, FLJ44854, IDN 3, IDN 3 protein, IDN 3 protein isoform A, IDN 3 protein isoform B, IDN 3B, IDN3 B, IDN3 protein, IDN3 protein isoform A, IDN3 protein isoform B, IDN3B, Mis 4, Mis4, Nipbl, NIPBL_HUMAN, Nipped B homolog Drosophila, Nipped B homolog, Nipped B like, Nipped B like protein, Nipped-B-like protein, Scc 2, SCC 2 homolog, Scc2, SCC2 homolog, Sister chromatid cohesion protein Mis4.

    Background: This gene encodes the homolog of the Drosophila melanogaster Nipped-B gene product and fungal Scc2-type sister chromatid cohesion proteins. The Drosophila protein facilitates enhancer-promoter communication of remote enhancers and plays a role in developmental regulation. It is also homologous to a family of chromosomal adherins with broad roles in sister chromatid cohesion, chromosome condensation, and DNA repair. The human protein has a bipartite nuclear targeting sequence and a putative HEAT repeat. Condensins, cohesins and other complexes with chromosome-related functions also contain HEAT repeats. Mutations in this gene result in Cornelia de Lange syndrome, a disorder characterized by dysmorphic facial features, growth delay, limb reduction defects, and mental retardation. Two transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Jul 2008].

    Pathways
    Sensory Perception of Sound, Stem Cell Maintenance
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