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NIPBL antibody (AA 2651-2805)

NIPBL Reactivity: Rat IF (cc), IF (p), IHC (p), IHC (fro), ICC Host: Rabbit Polyclonal unconjugated
Catalog No. ABIN1714202
  • Target See all NIPBL Antibodies
    NIPBL (Nipped-B like Protein (NIPBL))
    Binding Specificity
    • 14
    • 5
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    AA 2651-2805
    Reactivity
    • 15
    • 15
    • 5
    Rat
    Host
    • 24
    • 3
    • 2
    • 1
    Rabbit
    Clonality
    • 26
    • 4
    Polyclonal
    Conjugate
    • 12
    • 3
    • 2
    • 2
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    This NIPBL antibody is un-conjugated
    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)
    Cross-Reactivity
    Rat
    Predicted Reactivity
    Human,Mouse,Dog,Cow,Sheep,Pig,Horse,Chicken
    Purification
    Purified by Protein A.
    Immunogen
    KLH conjugated synthetic peptide derived from human IDN3
    Isotype
    IgG
  • Application Notes
    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' Badge
    by
    Gianluca Zambanini, Anna Nordin and Claudio Cantù; Cantù Lab, Gene Regulation during Development and Disease, Linköping University
    No.
    #104411
    Date
    04/26/2023
    Antigen
    NIPBL
    Lot Number
    AG10167864
    Method validated
    Cleavage Under Targets and Release Using Nuclease
    Positive Control

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

    Negative Control

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

    Notes

    Passed. The anti-NIPBL ABIN1714202 allows for CUT&RUN targeted profiling of NIPBL binding in mouse forelimb cells.

    'Independent Validation' Badge
    Validation Images
    Full Methods
    Primary Antibody
    ABIN1714202
    Secondary Antibody
    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.
    Experimental 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.
    Preservative
    ProClin
    Precaution of Use
    This product contains ProClin: a POISONOUS AND HAZARDOUS SUBSTANCE, which should be handled by trained staff only.
    Storage
    4 °C,-20 °C
    Storage Comment
    Shipped at 4°C. Store at -20°C for one year. Avoid repeated freeze/thaw cycles.
    Expiry Date
    12 months
  • Target
    NIPBL (Nipped-B like Protein (NIPBL))
    Alternative Name
    IDN3 (NIPBL Products)
    Synonyms
    CDLS antibody, CDLS1 antibody, IDN3 antibody, IDN3-B antibody, Scc2 antibody, SCC2W antibody, scc2 antibody, nipbl antibody, scc2-2 antibody, NIPBL antibody, nipbla antibody, wu:fi33d08 antibody, 4921518A06Rik antibody, 4933421G18Rik antibody, C79399 antibody, Idn3 antibody, NIPBL, cohesin loading factor antibody, Nipped-B homolog-like antibody, NIPBL, cohesin loading factor S homeolog antibody, nipped-B homolog b (Drosophila) antibody, nipped-B-like protein A antibody, Nipped-B homolog (Drosophila) antibody, NIPBL antibody, NIPBLL antibody, nipbl.S antibody, LOC427439 antibody, nipblb antibody, LOC100164947 antibody, nipbl antibody, Nipbl antibody
    Background

    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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