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TBX2/3 Antikörper

Reaktivität: Human, Maus, Ratte WB, IF, ICC Wirt: Kaninchen Polyclonal unconjugated
Produktnummer ABIN6265491
  • Target
    TBX2/3
    Reaktivität
    • 10
    • 10
    • 10
    • 1
    • 1
    • 1
    • 1
    Human, Maus, Ratte
    Wirt
    • 10
    Kaninchen
    Klonalität
    • 10
    Polyklonal
    Konjugat
    • 3
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    Unkonjugiert
    Applikation
    Western Blotting (WB), Immunofluorescence (IF), Immunocytochemistry (ICC)
    Spezifität
    TBX 2/3 Antibody detects endogenous levels of total TBX 2/3.
    Kreuzreaktivität
    Human, Maus, Ratte
    Homologie
    Pig(100%), Bovine(100%), Horse(100%), Sheep(100%), Rabbit(100%), Dog(100%), Chicken(100%), Xenopus(100%)
    Aufreinigung
    The antiserum was purified by peptide affinity chromatography using SulfoLinkTM Coupling Resin (Thermo Fisher Scientific).
    Isotyp
    IgG
  • Applikationshinweise
    WB: 1:500~1:3000, IF/ICC 1:100-1:500
    Beschränkungen
    Nur für Forschungszwecke einsetzbar
  • Validierung #104234 (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.
    #104234
    Datum
    01.03.2021
    Antigen
    TBX3
    Chargennummer
    10H2885
    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. ABIN6265491 allows for TBX3 targeted digestion using CUT&RUN.

    'Independent Validation' Siegel
    Validierungsbilder
    Protokoll
    Primärantikörper
    ABIN6265491
    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 antibody (Anti TBX3 (ABIN6265491), positive control, and negative control) to the respective tube, 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

    Peaks generated using ABIN6265491 for CUT&RUN aligned well with TBX3 ChIP-seq tracks from the same tissue (Zimmerli et. al., 2020).

  • Format
    Liquid
    Konzentration
    1 mg/mL
    Buffer
    Rabbit IgG in phosphate buffered saline ,  pH 7.4, 150  mM NaCl, 0.02 % sodium azide and 50 % glycerol.
    Konservierungsmittel
    Sodium azide
    Vorsichtsmaßnahmen
    This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
    Lagerung
    -20 °C
    Informationen zur Lagerung
    Store at -20 °C.Stable for 12 months from date of receipt
    Haltbarkeit
    12 months
  • Target
    TBX2/3
    Hintergrund

    Description: Transcriptional repressor involved in developmental processes. Probably plays a role in limb pattern formation. Acts as a negative regulator of PML function in cellular senescence.

    Molekulargewicht
    79kDa
    Gen-ID
    6926
    UniProt
    O15119, Q13207
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