RAD21 antibody (Center)

Catalog No. ABIN2856242

Product Details anti-RAD21 Antibody

Target: RAD21 (RAD21 Homolog (RAD21))

Binding Specificity: Center

Reactivity: Human, Mouse

Host: Rabbit

Clonality: Polyclonal

Conjugate: This RAD21 antibody is un-conjugated

Application: Western Blotting (WB), Immunofluorescence (IF), Immunoprecipitation (IP), Immunohistochemistry (Paraffin-embedded Sections) (IHC (p)), Immunocytochemistry (ICC)

Characteristics: Rabbit Polyclonal antibody to RAD21 (RAD21 homolog (S. pombe))
RAD21 antibody

Purification: Purified by antigen-affinity chromatography.

Immunogen: Recombinant protein encompassing a sequence within the center region of human RAD21. The exact sequence is proprietary.

Isotype: IgG

Application Details

Application Notes: Suggested dilution Reference ICC/IF 1:100-1:1000* IHC (Formalin-fixed paraffin-embedded sections) 1:100-1:1000* Immunoprecipitation 1:100-1:500* Western blot 1:500-1:3000* Not tested in other applications. *Optimal dilutions/concentrations should be determined by the researcher.Suggested dilutionReferenceICC/IF1:100-1:1000* IHC (Formalin-fixed paraffin-embedded sections)1:100-1:1000* Immunoprecipitation1:100-1:500* Western blot1:500-1:3000*

Comment:

Positive Control: K562 , THP-1 , HL-60 , NIH-3T3 , JC , BCL-1

Restrictions: For Research Use only

Independent Validation (CUT&RUN)

Validation #104616 (Cleavage Under Targets and Release Using Nuclease)

by: Cantù Lab, Gene Regulation during Development and Disease, Linköping University

No.: #104616

Date: 12/06/2024

Antigen: RAD21

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.

Validation Images

Library profiles comparing fragment size distributions on an E-Gel EX 2% agarose gel (Thermo Fisher). Fragments obtained from CUT&RUN using RAD21 ABIN2856242 (right) after library preparation, compared to the E-Gel Sizing DNA Ladder (Thermo Fisher) (left).

1. Alignment tracks from CTCF CUT&RUN in HEK293T cells. 2. Alignment tracks from CUT&RUN targeting RAD21 in HEK293T cells using ABIN2856242 antibody showing the FBRS locus. 3. RefSeq Genes.

Full Methods

Primary Antibody: ABIN2856242

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 ABIN2856242, 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.

Handling

Format: Liquid

Concentration: 1 mg/mL

Buffer: 1XPBS, 20 % Glycerol ( pH 7). 0.025 % ProClin 300 was added as a preservative.

Preservative: ProClin

Precaution of Use: This product contains ProClin: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.

Storage: -20 °C

Storage Comment: Keep as concentrated solution. Aliquot and store at -20°C or below. Avoid multiple freeze-thaw cycles.

References for anti-RAD21 antibody (ABIN2856242)

Ochiai, Hayashi, Umeda, Yoshimura, Harada, Shimizu, Nakano, Saitoh, Liu, Yamamoto, Okamura, Ohkawa, Kimura, Nikaido: "Genome-wide kinetic properties of transcriptional bursting in mouse embryonic stem cells." in: Science advances, Vol. 6, Issue 25, pp. eaaz6699, (2020) (PubMed).

Target Details for RAD21

Target: RAD21 (RAD21 Homolog (RAD21))

Alternative Name: RAD21 (RAD21 Products)

Background: The protein encoded by this gene is highly similar to the gene product of Schizosaccharomyces pombe rad21, a gene involved in the repair of DNA double-strand breaks, as well as in chromatid cohesion during mitosis. This protein is a nuclear phospho-protein, which becomes hyperphosphorylated in cell cycle M phase. The highly regulated association of this protein with mitotic chromatin specifically at the centromere region suggests its role in sister chromatid cohesion in mitotic cells.

Cellular Localization: Nucleus

Molecular Weight: 72 kDa

Gene ID: 5885

Pathways: Positive Regulation of Endopeptidase Activity