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Recombinant RAD21 antibody (N-Term)

RAD21 Reactivity: Human, Mouse WB, IF Host: Mouse Monoclonal unconjugated Recombinant Antibody
Catalog No. ABIN6731038
  • Target See all RAD21 Antibodies
    RAD21 (RAD21 Homolog (RAD21))
    Antibody Type
    Recombinant Antibody
    Binding Specificity
    • 16
    • 12
    • 4
    • 2
    • 2
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    N-Term
    Reactivity
    • 54
    • 29
    • 29
    • 6
    • 5
    • 5
    • 5
    • 4
    • 3
    • 3
    • 2
    • 2
    • 2
    • 2
    • 1
    Human, Mouse
    Host
    • 49
    • 3
    • 1
    Mouse
    Clonality
    • 46
    • 8
    Monoclonal
    Conjugate
    • 32
    • 2
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    This RAD21 antibody is un-conjugated
    Application
    • 46
    • 16
    • 13
    • 13
    • 10
    • 10
    • 9
    • 7
    • 6
    • 5
    • 4
    • 2
    • 1
    • 1
    • 1
    Western Blotting (WB), Immunofluorescence (IF)
    Brand
    AbFlex®
    Purification
    Protein A Chromatography
    Immunogen
    This antibody was raised against recombinant protein corresponding to the N-terminal half of mouse Rad21.
    Isotype
    IgG2a
    Top Product
    Discover our top product RAD21 Primary Antibody
  • Application Notes

    Validated Applications:
    IF: 0.25 - 1 µg/ml
    WB: 0.5- 2 μg/mL
    Comment

    AbFlex® antibodies are recombinant antibodies (rAbs) that have been generated using defined DNA sequences to produce highly specific, reproducible antibodies. Each AbFlex antibody contains a 6XHis tag, an avidin tag sequence for enzymatic biotin conjugation using the biotin ligase, BirA, and a sortase recognition motif (LPXTG) to attach a variety of labels directly to the antibody including fluorophores, enzymatic substrates (HRP, AP), peptides, drugs as well as solid supports. AbFlex Rad21 antibody was expressed as full-length IgG with mouse immunoglobulin heavy and light chains (IgG2a isotype) in mammalian 293 cells

    Restrictions
    For Research Use only
  • Validation #104617 (Cleavage Under Targets and Release Using Nuclease)
    'Independent Validation' Badge
    by
    Cantù Lab, Gene Regulation during Development and Disease, Linköping University
    No.
    #104617
    Date
    12/06/2024
    Antigen
    RAD21
    Lot Number
    Method validated
    Cleavage Under Targets and Release Using Nuclease
    Positive Control
    Anti H3K4me (antibodies-online, ABIN3023251)
    Negative Control
    Guinea Pig anti-rabbit IgG (antibodies-online, ABIN101961)
    Notes

    Passed.

    'Independent Validation' Badge
    Validation Images
    Full Methods
    Primary Antibody
    ABIN6731038
    Secondary Antibody
    Full Protocol
    • Cell harvest and nuclear extraction
      • Harvest 250,000 HEK293T cells per antibody
      • Centrifuge cell solution 5 min at 600 x g at RT.
      • Remove the liquid carefully.
      • Gently resuspend cells in 2 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 40 µL NE Buffer per sample.
    • Concanavalin A beads preparation
      • Prepare one 2 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 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 44ul binding buffer
    • Nuclei immobilization – binding to Concanavalin A beads
      • Carefully vortex the nuclei suspension and add 44 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 2 mL tube on the magnet stand 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, 2mM EDTA)
      • Incubate 5 min at RT.
      • Place the tube on the magnet stand and when the liquid is clear remove the supernatant.
      • Resuspend the beads in 200µl of wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, Roche Complete Protease Inhibitor EDTA-free) x each sample.
    • Primary antibody binding
      • Divide nuclei suspension into separate 200 µL PCR tubes, one for each antibody.
      • Add 2 µL antibody (RAD21 ABIN6731038, anti-H3K4me positive control ABIN3023251, and guinea pig anti-rabbit IgG negative control antibody ABIN101961) to the respective tube, corresponding to a 1:100 dilution.
      • Incubate at 4 °C ON.
      • 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 five times for a total of six washes.
    • pAG-MNase Binding
      • Prepare a 1.5 mL microcentrifuge tube containing 200 µL of pAG mix per 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 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 five times for a total of six washes.
      • Resuspend in 100 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 102 µl of 2 mM CaCl2 mix per sample (100 µl Wash Buffer + 2 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 100 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 remove the supernatant.
      • Resuspend the sample in 50 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 1h at 4°C.
      • Transfer the supernatant containing the pAG-MNase-bound digested chromatin fragments to fresh 200 µl PCR tubes.
    • DNA Clean up (Mag-Bind® TotalPure NGS - M1378-01)
      • 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 beads (Important!!! Do NOT resuspend the beads or remove the tubes from the magnet stand or the sample will be lost).
      • 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 µlL of beads for each sample).
      • Resuspend the beads + 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), 36bp PE.
    • Peak calling
      • Trim reads using using bbTools bbduk (BBMap - Bushnell B. - sourceforge.net/projects/bbmap/) to remove adapters, artifacts and repeat sequences.
      • Aligned reads were mapped to the hg38 human 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
  • Format
    Liquid
    Buffer
    Purified IgG in 140 mM Hepes, pH 7.5, 70 mM NaCl, 32 mM NaOAc, 0.035 % sodium azide, 30 % glycerol.
    Preservative
    Sodium azide
    Precaution of Use
    This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
    Storage
    -20 °C
  • Target
    RAD21 (RAD21 Homolog (RAD21))
    Alternative Name
    Rad21 (RAD21 Products)
    Molecular Weight
    100 kDa
    Pathways
    Positive Regulation of Endopeptidase Activity
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