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TBX3 Antikörper

TBX3 Reaktivität: Human, Maus, Ratte WB, ELISA, ICC, IF, CUT&RUN Wirt: Kaninchen Polyclonal unconjugated
Produktnummer ABIN6265491
  • Target Alle TBX3 Antikörper anzeigen
    TBX3 (T-Box 3 (TBX3))
    Reaktivität
    • 56
    • 21
    • 17
    • 6
    • 6
    • 5
    • 5
    • 5
    • 3
    • 2
    • 2
    • 2
    Human, Maus, Ratte
    Wirt
    • 57
    • 6
    • 1
    Kaninchen
    Klonalität
    • 58
    • 6
    Polyklonal
    Konjugat
    • 36
    • 10
    • 4
    • 4
    • 2
    • 2
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    Dieser TBX3 Antikörper ist unkonjugiert
    Applikation
    • 49
    • 34
    • 10
    • 7
    • 6
    • 5
    • 5
    • 3
    • 2
    • 1
    • 1
    • 1
    • 1
    Western Blotting (WB), ELISA, Immunocytochemistry (ICC), Immunofluorescence (IF), Cleavage Under Targets and Release Using Nuclease (CUT&RUN)
    Spezifität
    TBX3 antibody detects endogenous levels of total TBX3
    Kreuzreaktivität
    Human, Maus, Ratte (Rattus)
    Aufreinigung
    The antiserum was purified by peptide affinity chromatography using SulfoLinkTM Coupling Resin (Thermo Fisher Scientific).
    Immunogen
    A synthesized peptide derived from human TBX3
    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
    TBX3 (T-Box 3 (TBX3))
    Andere Bezeichnung
    TBX3 (TBX3 Produkte)
    Synonyme
    xtbx3 antikoerper, ums antikoerper, xhl antikoerper, tbx3-iso antikoerper, TBX3 antikoerper, TBX3-ISO antikoerper, UMS antikoerper, XHL antikoerper, D5Ertd189e antikoerper, T-box 3 antikoerper, T-box 3 S homeolog antikoerper, TBX3 antikoerper, tbx3.S antikoerper, tbx3 antikoerper, Tbx3 antikoerper
    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.

    Gene: TBX3

    Molekulargewicht
    79kDa
    Gen-ID
    6926
    UniProt
    O15119, Q13207
    Pathways
    Hormone Transport, Stem Cell Maintenance, Regulation of Muscle Cell Differentiation, Skeletal Muscle Fiber Development
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