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CUT&RUN Positive Control

Produktnummer ABIN6923144
  • Applikation
    Cleavage Under Targets and Release Using Nuclease (CUT&RUN), Cleavage Under Targets and Tagmentation (CUT&Tag)
    Verwendungszweck
    CUT&RUN Positive Control antibody of our CUT&RUN Sets.
    Spezifität
    This antibody reacts to Histone H3 trimethylated at Lysine 27 (K27me3).
    Kreuzreaktivität (Details)
    No cross reactivity with monomethylated Lysine 27 (K27me1) or dimethylated Lysine 27 (K27me2), or other methylations in histone H3.
    Produktmerkmale
    Rabbit monoclonal to Trimethylated Histone H3 Lysine 27.
    Aufreinigung
    Protein A affinity purified from an animal origin-free culture supernatant
  • Applikationshinweise
    Optimal working dilution should be determined by the investigator.
    Beschränkungen
    Nur für Forschungszwecke einsetzbar
  • Validierung #104231 (Cleavage Under Targets and Release Using Nuclease)
    'Independent Validation' Siegel
    by
    Mattias Pernebrink and Claudio Cantù; Cantù Lab, Gene Regulation during Development and Disease, Linköping University
    No.
    #104231
    Datum
    01.03.2021
    Antigen
    H3K27me3
    Chargennummer
    s-08-02482
    Validierte Anwendung
    Cleavage Under Targets and Release Using Nuclease
    Positivkontrolle
    Recombinant anti-H3K27me3 CUT&RUN Positive Control antibody (antibodies-online, ABIN6923144)
    Negativkontrolle
    Monoclonal anti-FLAG (Sigma-Aldrich, F3165)
    Bewertung

    Passed. ABIN6923144 serves as H3K27me3 positive control antibody in CUT&RUN.

    'Independent Validation' Siegel
    Validierungsbilder
    Protokoll
    Primärantikörper
    ABIN6923144
    Sekundärantikörper
    Full Protocol
    • Cell harvest
      • Harvest cells from day 10.5 mouse embryo front limbs, estimating 90,000 cells per antibody to be used at RT.
      • Centrifuge cell solution 3 min at 600 x g at RT.
      • Remove the liquid carefully.
      • Gently resuspend cells in 1 mL Wash Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, Roche Complete Protease Inhibitor EDTA-free) by pipetting and transfer cell solution to a 2 mL microcentrifuge tube.
      • Centrifuge cell solution 3 min at 600 x g at RT and discard the supernatant.
      • Repeat twice for a total of three washes.
      • Resuspend cell pellet in 1 mL Wash Buffer by gently pipetting.
    • Concanavalin A beads preparation
      • Prepare one 1.5 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 cell suspension and add 10 µL of the Con A beads in Binding Buffer to the cell suspension for each sample.
      • Close tube tightly and rotate for 10 min at RT.
    • Cell permeabilization and primary antibody binding
      • Divide cell suspension into separate 2 mL microcentrifuge tubes, one for each antibody (500,000 cells per sample).
      • Place the microcentrifuge tubes on a magnetic stand until the fluid is clear. Remove the liquid carefully.
      • Remove the microcentrifuge tubes from the magnetic stand.
      • Place each tube at a low angle on the vortex mixer set to a low speed and add 150 µL Digitonin Wash buffer (wash buffer with 0.025% (wt/vol) Digitonin) supplemented with 2 mM EDTA.
      • Gently vortex the microcentrifuge tubes until the beads are resuspended.
      • Add 1.5 µL rabbit anti-H3K27me3 antibody CUT&RUN positive control (antibodies-online, ABIN6923144, lot s-08-02482) corresponding to a 1:100 dilution.
      • Rotate the microcentrifuge tubes ON at 4 °C.
      • Spin down the liquid and place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
      • Remove the microcentrifuge tubes from the magnetic stand.
      • Resuspend with 1 mL Digitonin Wash Buffer and mix by inversion. If clumping occurs, gently remove the clumps with a 1 ml pipette tip.
      • Repeat once for a total of two washes.
    • pA-MNase Binding
      • Place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
      • Remove the microcentrifuge tubes from the magnetic stand.
      • Vortex the sample at low speed and add 150 μL pA-MNase solution at 700 ng/mL (1:200 dilution of a 140 µg/mL glycerol stock in Digitonin Wash Buffer) per sample, gently resuspending the beads by pipetting.
      • Rotate the microcentrifuge tubes for 1 h at 4 °C.
      • Spin down the liquid and place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
      • Remove the microcentrifuge tubes from the magnetic stand.
      • Resuspend with 1 mL Digitonin Wash Buffer and mix by inversion. If clumping occurs, gently remove the clumps with a 1 mL pipette tip.
      • Repeat once for a total of two washes.
    • MNase digestion and release of pA-MNase-antibody-chromatin complexes
      • 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.
      • Place each tube at a low angle on the vortex mixer set to a low speed and add 100 μL Digitonin Wash buffer per sample along the side of the tube.
      • Place tubes in a heat block, kept on ice, and allow to chill.
      • Add 2 μL 0.1 M CaCl2 to each sample.
      • Incubate tubes at 0 °C for 30 min.
      • Add 100 μL 2xSTOP buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.05% (wt/vol) Digitonin, 100 μg/mL RNAse A, 50 μg/mL Glycogen).
      • Incubate tubes at 37 °C for 30 min.
      • Place the tubes on a magnet stand until the fluid is clear.
      • Transfer the supernatant containing the pA-MNase-bound digested chromatin fragments to fresh 1.5 mL microcentrifuge tubes.
    • DNA extraction
      • Add 2 µL 10% SDS to a final concentration of 0.1% and 2.5 µL Proteinase K (20 mg/mL) to each supernatant.
      • Gently vortex tubes at a low speed of approximately 1,100 rpm.
      • Incubate tubes at 50 °C for 1 h.
      • Add 200 µL PCI to tube.
      • Vortex tubes thoroughly at high speed until the liquid appears milky.
      • Centrifuge tubes in a tabletop centrifuge at 16,000 x g at RT for 5 min.
      • Carefully transfer to upper aqueous phase to a fresh 1.5 mL microcentrifuge tube containing 2 µL glycogen (diluted 1:10 to 2 mg/mL from the 20 mg/mL stock solution).
      • Add 20 µL 3 M NaOAc pH 5.2.
      • Add 400 µL 100% ethanol.
      • Place tubes for at -20 °C ON.
      • Centrifuge tubes in a tabletop centrifuge at 16,000 x g at 4 °C for 5min.
      • Remove the liquid carefully with a pipette.
      • Wash pellet with 1ml 70% ethanol.
      • Centrifuge tubes in a tabletop centrifuge at 16,000 x g at 4 °C for 1 min.
      • Remove the liquid carefully with a pipette.
      • Air-dry the pellet, then dissolve in 30 µL 1 mM Tris-HCl, 0.1 mM EDTA.
    • Library preparation and sequencing
      • Libraries were prepared using KAPA HyperPrep Kit using KAPA Dual-Indexed adapters according to protocol.
      • Samples were sequenced on an Illumina NextSeq 500 sequencer, using a NextSeq 500/550 High Output Kit v2.5 (75 Cycles), 36bp PE.
    • Peak calling
      • Reads were mapped to the GRCm38 (mm10) mouse genome using Bowtie2 with options: --local --very-sensitive- local --no-unal --no-mixed --no-discordant.
      • Peaks were called using MACS2 with options -f BAMPE --keep-dup all –nomodel.
    Anmerkungen

    PCUT&RUN libraries generated using the H3K27me3 CUT&RUN positive control antibody ABIN6923144 show the pattern typical for a nucleosome ladder.

  • Format
    Liquid
    Konzentration
    0.2 μg/μL
    Buffer
    50% Glycerol/PBS with 1% BSA and 0.09% Sodium azide
    Konservierungsmittel
    Sodium azide
    Vorsichtsmaßnahmen
    This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
    Lagerung
    4 °C
    Haltbarkeit
    12 months
  • Pagin, Pernebrink, Giubbolini, Barone, Sambruni, Zhu, Chiara, Ottolenghi, Pavesi, Wei, Cantù, Nicolis: "Sox2 controls neural stem cell self-renewal through a Fos-centered gene regulatory network." in: Stem cells (Dayton, Ohio), (2021) (PubMed).

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