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Rad21

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RAD21 is a protein that plays a role in the structural maintenance of chromosomes. It is a component of the cohesin complex, which is responsible for holding sister chromatids together during cell division.

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Lab products found in correlation

Histone h3, by Abcam (4 mentions) H3k4me2, by Abcam (3 mentions) H3k27ac, by Active Motif (3 mentions) Protease inhibitor cocktail, by Roche (3 mentions) Rad21, by Santa Cruz Biotechnology (2 mentions) Cenp f, by Novus Biologicals (2 mentions) Nipbl, by Santa Cruz Biotechnology (2 mentions) Anti rabbit igg hrp linked antibody, by Cell Signaling Technology (2 mentions) Proliferating cell nuclear antigen (pcna), by Santa Cruz Biotechnology (2 mentions) Alpha tubulin, by Merck Group (2 mentions) Western lightning plus ecl, by PerkinElmer (2 mentions) Chemidoc mp, by Bio-Rad (2 mentions) Mini protean tgx precast gel, by Bio-Rad (2 mentions) C myc, by Santa Cruz Biotechnology (2 mentions) Odyssey clx, by LI COR (2 mentions) β actin, by Santa Cruz Biotechnology (2 mentions) Gapdh, by Abcam (2 mentions) H3k4me3, by Abcam (2 mentions) Cd11b pe cyanine5, by Thermo Fisher Scientific (2 mentions) F4 80 fitc, by Thermo Fisher Scientific (2 mentions)

Spelling variants of this product

Anti-RAD21 (11 citations) RAD21 antibody (4 citations) Anti-RAD21 antibody (3 citations) Anti-Rad21 Ab (2 citations)

26 protocols using rad21

1

Immunofluorescence and ChIP Assay Protocols

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The following antibodies were used for immunofluorescence studies: rabbit anti-Daxx (Sigma, D7810), rabbit anti-ATRX H-300 (Santa Cruz, Sc15408), rabbit anti-PML (Bethyl, A301167A), rabbit anti-EZH2 (Cell Signaling, 4905S), rabbit anti-H3K27me3 (Active motif, 39155), mouse anti-ORC2 (MBL, M0553), and mouse anti-αTubulin/Alexa488 (Invitrogen, 322588). Secondary antibodies AlexaFluor488 or AlexaFluor594 were purchased from Invitrogen. The following antibodies were used for Western blotting: mouse anti-ORF50 (provided by Erle Robertson, UPENN), mouse anti-ORF45 (provided by Yan Yuan, UPENN), rat anti-LANA (Advanced Biotechnologies Inc., 13210), rabbit anti-Daxx (Sigma), and anti-actin-HRP (Sigma, A23852). Antibodies used in ChIP assay include: rabbit polyclonal antibodies to histone H3K4me3 (Millipore, 07473), histone H3K27me3 (Active motif,391155), total histone H3 (Bethyl), ORC2 (MBL, M0553), Rad21 (Abcam, ab992), CTCF (Millipore, 07729), or rabbit IgG (Santa Cruz Biotechnology, sc-2027), and rat polyclonal anti-LANA (Advanced Biotechnologies Inc.).
De Leo A., Deng Z., Vladimirova O., Chen H.S., Dheekollu J., Calderon A., Myers K.A., Hayden J., Keeney F., Kaufer B.B., Yuan Y., Robertson E, & Lieberman P.M. (2019). LANA oligomeric architecture is essential for KSHV nuclear body formation and viral genome maintenance during latency. PLoS Pathogens, 15(1), e1007489.
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2

Immunofluorescence and Western Blot Protocols

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Immunofluorescence was performed using the following primary antibodies: RAD21 (Santa Cruz sc-166973, 1:100), PCNA (Santa Cruz sc-56, 1:100), CENPF (Novus Biologicals NB500–101, 1:100). Secondary antibodies used: Goat anti-Rabbit (Jackson ImmunoResearch 111-165-003, 1:200), Sheep anti-Mouse (Jackson ImmunoResearch 505-605-003, 1:100).
Western blots were performed with the following primary antibodies: RAD21 (Abcam ab992, 1:500 or 1:1000), NIPBL (Santa Cruz sc-374625, 1:400), WAPL (Santa Cruz sc-365189, 1:250), alpha tubulin (Sigma T6074, 1:1,000), Histone H3 (Abcam ab1791, 1:40,000), and CTCF (Santa Cruz sc-271474, 1:500). Secondary antibodies used: Goat anti-Rabbit (Jackson ImmunoResearch 111-165-003, 1.3:7,000 - 1.3:10,000), Goat anti-Mouse (Jackson ImmunoResearch 115-545-003, 1.3:7,000 - 1.3:10,000), Anti-mouse IgG HRP-linked Antibody (Cell Signaling Technologies #7076, 1:5,000), Anti-rabbit IgG HRP-linked Antibody (Cell Signaling Technologies #7074, 1:5,000).
Luppino J.M., Park D.S., Nguyen S.C., Lan Y., Xu Z., Yunker R, & Joyce E.F. (2020). Cohesin promotes stochastic domain intermingling to ensure proper regulation of boundary-proximal genes. Nature genetics, 52(8), 840-848.
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3

Covalent Crosslinking of Proteins to Nucleic Acids

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To covalently crosslink the proteins to nucleic acids, glioma cells (2 × 107) were subjected to UV irradiation (200 mJ/cm2). The cells were then lysed with RIPA lysis buffer containing Protease Inhibition Cocktail and RNase Inhibitor. The immunoglobin-coated protein magnetic beads (Thermofisher scientific) were incubated with CTCF (D31H2) XP Rabbit mAb (#3418) and Rad21 (ab992, Abcam) antibodies to immunopurify the respective complexes. The complexes were washed with RIPA buffer, and the samples treated with DNAse I (Promega) for 30 min at 37°C followed by proteinase K (10% SDS and 10 mg/ml proteinase K in RIPA buffer) for 30 min at 37°C with shaking. RNA was further isolated using phenol:chloroform:isoamyl alcohol (25:24:1) solution, precipitated using isopropanol, and re-suspended in RNase-free water. qRT-PCR was performed for nascent HOXD-AS2 and LINC01116 transcripts. Primers are listed in Supplementary Table 2. Fold enrichment over IgG was calculated in two steps: 1) DDCt = (Ct IP) - (Ct IgG); 2) (2^-DDCt).
Deforzh E., Uhlmann E.J., Das E., Galitsyna A., Arora R., Saravanan H., Rabinovsky R., Wirawan A.D., Teplyuk N.M., Fatimy R.E., Perumalla S., Jairam A., Wei Z., Mirny L, & Krichevsky A.M. (2022). Promoter and enhancer RNAs regulate chromatin reorganization and activation of miR-10b/HOXD locus, and neoplastic transformation in glioma. Molecular cell, 82(10), 1894-1908.e5.
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4

Pluripotency Marker Expression Analysis

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ESCs were lysed in 1xSDS sampling buffer (200mM Tris HCl pH7, 10% glycerol, 2% SDS, 4% beta-mercaptoethanol, 400mM DTT, 0.4% bromophenol blue) preheated at 95°C. Lysates were further sonicated and denatured at 95°C for 5min. Proteins from each sample was resolved by SDS-PAGE using Mini-PROTEAN® TGX™ Precast Gels (Bio-Rad). Primary antibodies used include: CTCF (Millipore, 07729), RAD21 (abcam, 154769), OCT4 (Santa Cruz, sc-5279), SOX2 (Millipore, AB5603), BRD2(Bethyl laboratories, A302-583A), BRD3(Active Motif, 61489), BRD4(Abcam, ab128874 for both short and long isoforms ) or BRD4 (Active Motif, 39010, for long isoform only). We used HRP conjugated secondary antibodies (Pierce) at a dilution of 1:3000. Western blot was exposed to Western Lightning Plus-ECL (PerkinElmer) and imaged in a ChemiDoc MP (Bio-Rad) detection system.
Xie L., Dong P., Qi Y., Hsieh T.H., English B.P., Jung S., Chen X., De Marzio M., Casellas R., Chang H.Y., Zhang B., Tjian R, & Liu Z. (2022). BRD2 Compartmentalizes the Accessible Genome. Nature genetics, 54(4), 481-491.
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5

Protein Expression and Cytokine Profiling

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RAD21 (Abcam ab992), β-Actin (Santa Cruz sc-69879), c-Myc (Santa Cruz sc-40 9E10) and GAPDH (Abcam ab8245) were used for immunoblots. Cytokine arrays (R&D ARY006) and IFN-β ELISA (RnD 42400-1) were performed following manufacturer’s instructions using supernatant from macrophages collected 8 h after LPS stimulation (10 ng/ml). Immunoblots and antibody arrays were imaged using an Odyssey CLx instrument (LI-COR).
Cuartero S., Weiss F.D., Dharmalingam G., Guo Y., Ing-Simmons E., Masella S., Robles-Rebollo I., Xiao X., Wang Y.F., Barozzi I., Djeghloul D., Amano M.T., Niskanen H., Petretto E., Dowell R.D., Tachibana K., Kaikkonen M.U., Nasmyth K.A., Lenhard B., Natoli G., Fisher A.G, & Merkenschlager M. (2018). Control of inducible gene expression links cohesin to hematopoietic progenitor self-renewal and differentiation. Nature immunology, 19(9), 932-941.
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6

Chromatin Fractionation and Western Blot Analysis

Cited 1 time
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For western blot, equal amounts of live cells that were non-treated or treated with IAA for 6 hours and then subject to STI-571 treatment for 4-days were collected and processed for chromatin fractionation. In brief, cells were lysed in Buffer 1 (100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 10 mM PIPES, pH 6.8, 1 mM EGTA, and 0.2% Triton X-100) containing 1x protease cocktail inhibitors (Roche, #11836170001) and incubated on-ice for 5 min. After centrifugation, the pellet was gently washed three times in Buffer 1, resuspended in Buffer 2 (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, and 0.1% SDS) containing protease cocktail inhibitors, and then sonicated. Nuclear extracts were resolved on 10–12% (v/v) SDS-PAGE. Immunoblotting was performed according to standard procedures with antibodies as follows: Rad21 (Abcam, #ab992), CTCF (Millipore, #07–729), V5 (Invitrogen, #R960–25), Histone H3 (Abcam, #ab176842), GFP (Takara Bio Inc., #632380) and RAG254 (link).
Ba Z., Lou J., Ye A.Y., Dai H.Q., Dring E.W., Lin S.G., Jain S., Kyritsis N., Kieffer-Kwon K.R., Casellas R, & Alt F.W. (2020). CTCF orchestrates long-range cohesin-driven V(D)J recombinational scanning. Nature, 586(7828), 305-310.
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7

ChIP-seq Profiling of Transcription Factors

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ChIP-seq experimentation was performed as previously outlined (Reddy et al. 2009 (link)). We utilized Flag (Sigma, F1804), RAD21 (abcam, ab992), CREB1 (Santa Cruz Biotechnology, sc-240), and GABPA (Santa Cruz Biotechnology, sc-28312) antibodies for all ChIP-seq experiments. ChIP-seq libraries were run on an Illumina HiSeq 2500 next-generation sequencer. Technical replicates were done with independent chromatin immunoprecipitation experimentations from the same transfected cell pool. Biological replicates represent independent cellular transfection experiments.
Savic D., Partridge E.C., Newberry K.M., Smith S.B., Meadows S.K., Roberts B.S., Mackiewicz M., Mendenhall E.M, & Myers R.M. (2015). CETCh-seq: CRISPR epitope tagging ChIP-seq of DNA-binding proteins. Genome Research, 25(10), 1581-1589.
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8

ChIP-qPCR Assay Protocol

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ChIP experiment was performed as previously reported [33 (link)]. Cells were fixed and sheared by using Covaris M200 (Covaris, Woburn, MA, USA). The fragmented chromatin was incubated with the following primary Abs; KLF5 (Abcam, Cambridge, UK), MEIS1 (Abcam), RHOXF1 (GeneTex, Irvine, CA, USA), ZNF354C (Abcam), BRD4 (Bethyl Laboratories, Montgomery, TX, USA), MED1 (Bethyl Laboratories), RAD21 (Abcam). The Ab against normal rabbit IgG (Cell Signaling Technology, Danvers, MA, USA) was used as a negative control. The purified DNA was subjected to qPCR. qPCR was performed as described in the qRT-PCR section. The sequences of the primers are listed in Supplementary Table S1.
Takeda T., Yokoyama Y., Takahashi H., Okuzaki D., Asai K., Itakura H., Miyoshi N., Kobayashi S., Uemura M., Fujita T., Ueno H., Mori M., Doki Y., Fujii H., Eguchi H, & Yamamoto H. (2021). A stem cell marker KLF5 regulates CCAT1 via three-dimensional genome structure in colorectal cancer cells. British Journal of Cancer, 126(1), 109-119.
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9

SWI/SNF Subunit Interactions in Cells

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Cells and mice tissues were lysed in ice-cold protein extraction buffer [50 mM tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 50 mM NaF, 10 mM sodium pyrophosphate, 10 mM sodium β-glycerophosphate] with protease inhibitor cocktails (Roche) and quantified using the BCA Protein Assay Kit (ThermoFisher). The lysates were immunoblotted against primary antibodies as follows: ARID1A (Cell Signaling Technology, 12354S), BRG1 (Santa Cruz Biotechnology, sc-17796), BAF170 (Bethyl Laboratories, A301-039A), BAF53A (Bethyl Laboratories, A301-391A), SNF5 (Santa Cruz Biotechnology, sc-13055), CTCF (Cell Signaling Technology, 3418S), RAD21 (Abcam, ab992), SMC3 (Abcam, ab9263), HA tag (Abcam, ab18181), Flag tag (Abcam, ab49763), T7 tag (Abcam, ab9138), PMP22 (Santa Cruz Biotechnology, sc-515199), GSC (R&D Systems, AF4086) and β-actin (Sigma, A5441). The blots were visualized with peroxidase-coupled secondary antibodies. Co-immunoprecipitation (Co-IP) assay was conducted with the same antibodies. Briefly, nuclear extracts were diluted with lysis buffer to a final concentration of 1 mg/ml and incubated with 2 µg of antibody on a rotator overnight. Protein G beads (Millipore) were added and incubated for 3 h. Then the beads were washed with 1 ml hypersaline lysis buffer (300 mM NaCl) for 5 times. Finally, beads were resuspended in SDS loading buffer and analyzed by immunoblotting.
Shang X.Y., Shi Y., He D.D., Wang L., Luo Q., Deng C.H., Qu Y.L., Wang N, & Han Z.G. (2021). ARID1A deficiency weakens BRG1-RAD21 interaction that jeopardizes chromatin compactness and drives liver cancer cell metastasis. Cell Death & Disease, 12(11), 990.
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10

Protein Expression and Cytokine Profiling

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RAD21 (Abcam ab992), β-Actin (Santa Cruz sc-69879), c-Myc (Santa Cruz sc-40 9E10) and GAPDH (Abcam ab8245) were used for immunoblots. Cytokine arrays (R&D ARY006) and IFN-β ELISA (RnD 42400-1) were performed following manufacturer’s instructions using supernatant from macrophages collected 8 h after LPS stimulation (10 ng/ml). Immunoblots and antibody arrays were imaged using an Odyssey CLx instrument (LI-COR).
Cuartero S., Weiss F.D., Dharmalingam G., Guo Y., Ing-Simmons E., Masella S., Robles-Rebollo I., Xiao X., Wang Y.F., Barozzi I., Djeghloul D., Amano M.T., Niskanen H., Petretto E., Dowell R.D., Tachibana K., Kaikkonen M.U., Nasmyth K.A., Lenhard B., Natoli G., Fisher A.G, & Merkenschlager M. (2018). Control of inducible gene expression links cohesin to hematopoietic progenitor self-renewal and differentiation. Nature immunology, 19(9), 932-941.
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