Anti-Rad21 抗体 - ChIP Grade (ab992)

Reviews (18)Q&A (9)References (107)

製品の概要

  • 製品名

    Anti-Rad21 antibody - ChIP Grade
    Rad21 一次抗体 製品一覧

  • 製品の詳細

    Rabbit polyclonal to Rad21 - ChIP Grade

  • 由来種

    Rabbit

  • 特異性

    The epitope recognized by ab992 maps to a region between residue 575 and the C-terminus (residue 631) human Rad21 homolog using the numbering given in entry NP_006256.1 (GeneID 5885).

  • アプリケーション

    適用あり: IHC-P, ChIP, ICC/IF, WB, IPmore details

  • 種交差性

    交差種: Mouse, Human, Xenopus laevis, Indian muntjac
    交差が予測される動物種: Rat, Rabbit, Horse, Chicken, Guinea pig, Cow, Dog, Turkey, Chimpanzee, Gorilla, Chinese hamster, Orangutan, Elephant

  • 免疫原

    Synthetic peptide (Human) conjugated to KLH - which represented a portion of human Rad21 encoded within exon 14 (LocusLink ID 5885).

  • ポジティブ･コントロール

    • HeLa cell lysate. FFPE human breast fibroadenoma tissue sections.

  • 特記事項

      

製品の特性

アプリケーション

Our Abpromise guarantee covers the use of ab992 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

アプリケーション Abreviews 特記事項
IHC-P Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
ChIP Use at an assay dependent concentration.
ICC/IF Use at an assay dependent concentration.

Used at a dilution of 1/200 for 30 min incubation (see Abreview for further information).
WB 1/500 - 1/5000. Predicted molecular weight: 72 kDa.

Band observed at ~130 kDa.
IP Use a concentration of 1 - 4 µg/ml.

ターゲット情報

  • 機能

    Cleavable component of the cohesin complex, involved in chromosome cohesion during cell cycle, in DNA repair, and in apoptosis. The cohesin complex is required for the cohesion of sister chromatids after DNA replication. The cohesin complex apparently forms a large proteinaceous ring within which sister chromatids can be trapped. At metaphase-anaphase transition, this protein is cleaved by separase/ESPL1 and dissociates from chromatin, allowing sister chromatids to segregate. The cohesin complex may also play a role in spindle pole assembly during mitosis. Also plays a role in apoptosis, via its cleavage by caspase-3/CASP3 or caspase-7/CASP7 during early steps of apoptosis: the C-terminal 64 kDa cleavage product may act as a nuclear signal to initiate cytoplasmic events involved in the apoptotic pathway.

  • 配列類似性

    Belongs to the rad21 family.

  • ドメイン

    The C-terminal part associates with the head of SMC1A, while the N-terminal part binds to the head of SMC3.

  • 翻訳後修飾

    Cleaved by separase/ESPL1 at the onset of anaphase. Cleaved by caspase-3 and caspase-7 at the beginning of apoptosis. The cleavage by ESPL1 and caspase-3 take place at different sites.
    Phosphorylated; becomes hyperphosphorylated in M phase of cell cycle. The large dissociation of cohesin from chromosome arms during prophase may be partly due to its phosphorylation by PLK.

  • 細胞内局在

    Nucleus. Chromosome. Chromosome > centromere. Associates with chromatin. Before prophase it is scattered along chromosome arms. During prophase, most of cohesin complexes dissociate from chromatin probably because of phosphorylation by PLK, except at centromeres, where cohesin complexes remain. At anaphase, it is cleaved by separase/ESPL1, leading to the dissociation of the complex from chromosomes, allowing chromosome separation. Once cleaved by caspase-3, the C-terminal 64 kDa cleavage product translocates to the cytoplasm, where it may trigger apoptosis.
  • Information by UniProt

  • 参照データベース

    see all

  • 別名

    • CDLS4 antibody
    • Double-strand-break repair protein rad21 homolog antibody
    • hHR21 antibody
    • HR21 antibody
    • HRAD21 antibody
    • KIAA0078 antibody
    • MCD1 antibody
    • Nuclear matrix protein 1 antibody
    • NXP-1 antibody
    • NXP1 antibody
    • Protein involved in DNA double-strand break repair antibody
    • RAD21 antibody
    • RAD21 homolog (S. pombe) antibody
    • RAD21 homolog antibody
    • RAD21_HUMAN antibody
    • Scc1 antibody
    • SCC1 homolog antibody
    see all

画像

  • Western blot - Anti-Rad21 antibody - ChIP Grade (ab992)
    Western blot - Anti-Rad21 antibody - ChIP Grade (ab992)
    Anti-Rad21 antibody - ChIP Grade (ab992) at 1/1000 dilution + HeLa whole cell lysate

    Developed using the ECL technique.

    Predicted band size: 72 kDa
    Observed band size: 75 kDa
    why is the actual band size different from the predicted?
    Additional bands at: 130 kDa (possible cross reactivity)

  • Immunocytochemistry/ Immunofluorescence - Anti-Rad21 antibody - ChIP Grade (ab992)
    Immunocytochemistry/ Immunofluorescence - Anti-Rad21 antibody - ChIP Grade (ab992)

    ab992 at a 1/200 dilution staining Asynchronous and paraformaldehyde-fixed (4%) HeLa cells by immunocytochemistry. The antibody was incubated with the cells 30 minutes and then detected using a Cy3 conjugated Goat Anti-Mouse IgG (H+L) antibody.

    This image is courtesy of an Abreview by Kirk McManus submitted on 27 February 2006.

    See Abreview

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Rad21 antibody - ChIP Grade (ab992)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Rad21 antibody - ChIP Grade (ab992)
    IHC image of Rad21 staining in human breast fibroadenoma formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab992, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
  • Western blot - Anti-Rad21 antibody - ChIP Grade (ab992)
    Western blot - Anti-Rad21 antibody - ChIP Grade (ab992)
    Anti-Rad21 antibody - ChIP Grade (ab992) at 1/2000 dilution + HeLa whole cell extract (extraction was achieved using RIPA buffer and 1% SDS) at 25 µg/ml

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab6721) at 1/1500 dilution

    Predicted band size: 72 kDa



    A rad21 knock down negative control was employed.

    This image is courtesy of an Abreview submitted on 9 September 2005. We do not have any further information relating to this image.

    See Abreview

  • Western blot - Anti-Rad21 antibody - ChIP Grade (ab992)
    Western blot - Anti-Rad21 antibody - ChIP Grade (ab992)
    All lanes : Anti-Rad21 antibody - ChIP Grade (ab992) at 2 µg/ml

    Lane 1 : HeLa at 50 µg
    Lane 2 : HeLa at 15 µg
    Lane 3 : HeLa at 5 µg
    Lane 4 : 293T at 50 µg
    Lane 5 : NIH3T3 at 50 µg

    Predicted band size: 72 kDa


    Exposure time: 30 seconds

参考文献

This product has been referenced in:

  • Amat R  et al. Rapid reversible changes in compartments and local chromatin organization revealed by hyperosmotic shock. Genome Res 29:18-28 (2019). Read more (PubMed: 30523037) »
  • Meisenberg C  et al. Repression of Transcription at DNA Breaks Requires Cohesin throughout Interphase and Prevents Genome Instability. Mol Cell 73:212-223.e7 (2019). Read more (PubMed: 30554942) »
See all 107 Publications for this product

ab992 を使用した論文を発表された方は、こちらまでお知らせください。データシートに掲載させていただきます。

レビューと Q&A

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レビューを送る 質問を送る

1-10 of 27 Abreviews or Q&A

Question

Customer kindly contacted us regarding positve and negative primers for our ChIP grade antibodies.

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Answer

I've compiled some information below regarding potential primer sets for the ChIP antibodies we talked about, where information was lacking (verified in ChIP by other researches), I included links to abReviews and references that used the antibody in ChIP.

1. ab817: GAPDH (+), GAD1 (-), AFM (-), Factor VIII (-)

2. ab1791: GAPDH, RPL30, ALDOA, MYO-D, SERPINA, AFM, SAT2, SATa (each batch is verified against this primer set, an example image of one batch test is given on the datasheet)

3. ab7030: was verified in ChIP by other researchers submitting data through our abReview system and specific references. Their positive and negative controls used are given through the link below, as well as a "click to contact this user" link that allows you to contact the researchers if you would like more specific primer information. The specific references with ChIP listed in bold are good sources for positive and negative primers as well.
a) Abreview: (NFKBIA promoter (+), GAPDH (-)) https://www.abcam.com/HDAC3-antibody-ChIP-Grade-ab7030-reviews.html?intabreviewid=25057
b) Abreview: https://www.abcam.com/HDAC3-antibody-ChIP-Grade-ab7030-reviews.html?intabreviewid=18386
c) Specific references: https://www.abcam.com/HDAC3-antibody-ChIP-Grade-ab7030-references.html

4. ab111069 Untested in ChIP.

5. ab992: was verified in ChIP by other researchers (abReviews and specific references)
a) Specific references: https://www.abcam.com/Rad21-antibody-ChIP-Grade-ab992-references.html
b) Abreview: https://www.abcam.com/Rad21-antibody-ChIP-Grade-ab992-reviews.html?intabreviewid=14542
c) Abreview: (EBV 3+ (+), EBV1,2,4,5,6 (-), GAPDH (-), Cen (-)) https://www.abcam.com/Rad21-antibody-ChIP-Grade-ab992-reviews.html?intabreviewid=33942

Our ChIP primer sets:
https://www.abcam.com/index.html?pageconfig=resource&rid=10600

Read More

ChIP abreview for Anti-Rad21 antibody - ChIP Grade

 Below Average
Abreviews
abreview image
Application
ChIP
Sample
Mouse Tissue lysate - nuclear (Limb buds, forebrain, ESC)
Negative control
We perform ChIP-seq
Specification
Limb buds, forebrain, ESC
Detection step
Other
Type
Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: 37% Formaldehyde (Sigma), dilu
Positive control
We perform ChIP-seq
Read More

Daniel Ibrahim

Verified customer

投稿 Nov 12 2018

Western blot abreview for Anti-Rad21 antibody - ChIP Grade

 Excellent
Abreviews
abreview image
Application
Western blot
Sample
Human Cell lysate - whole cell (Whole cell lysate resolved on a 12% SDS PAGE gel)
Gel Running Conditions
Reduced Denaturing (12% SDS gel)
Loading amount
25 µg
Treatment
HEK293T cells transfected with HA-Rad21 plasmid for overexpression
Specification
Whole cell lysate resolved on a 12% SDS PAGE gel
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Read More

Abcam user community

Verified customer

投稿 Jan 19 2017

Immunocytochemistry/ Immunofluorescence abreview for Anti-Rad21 antibody - ChIP Grade

 Good
Abreviews
abreview image
Application
Immunocytochemistry/ Immunofluorescence
Blocking step
Cells are incubated with primary antibody + BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: 37°C
Sample
Donkey Cell (Skin fibroblasts)
Specification
Skin fibroblasts
Permeabilization
Yes - 1% PFA + 0.2% Triton
Fixative
Paraformaldehyde
Read More

Abcam user community

Verified customer

投稿 Nov 12 2014

Question

What does this antibody recognize on human Rad21?

Read More
Answer

The epitope recognized by ab992 maps to a region between residue 575 and the C-terminus (residue 631) human Rad21 homolog using the numbering given in entry NP_006256.1 (GeneID 5885).

Read More

Immunocytochemistry/ Immunofluorescence abreview for Anti-Rad21 antibody - ChIP Grade

 Good
Abreviews
abreview image
Application
Immunocytochemistry/ Immunofluorescence
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 25°C
Sample
Mouse Cell (primary hepatoctyes)
Specification
primary hepatoctyes
Permeabilization
Yes - Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Fixative
Paraformaldehyde
Read More

Mr. Shengyun Ma

Verified customer

投稿 Oct 07 2013

Immunoprecipitation abreview for Anti-Rad21 antibody - ChIP Grade

 Below Average
Abreviews
abreview image
Application
Immunoprecipitation
Immuno-precipitation step
Protein A
Sample
Human Cell lysate - whole cell (Hep3B)
Specification
Hep3B
Total protein in input
30 µg
Read More

Abcam user community

Verified customer

投稿 Sep 16 2013

ChIP abreview for Anti-Rad21 antibody - ChIP Grade

 Good
Abreviews
abreview image
Application
ChIP
Detection step
Real-time PCR
Sample
Mouse Cell lysate - whole cell (primary hepatoctyes)
Specification
primary hepatoctyes
Negative control
IgG
Type
Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: 1% formaldehyde
Positive control
Suz12 on h19 ICR
Read More

Abcam user community

Verified customer

投稿 Sep 16 2013

Western blot abreview for Anti-Rad21 antibody - ChIP Grade

 Average
Abreviews
abreview image
Application
Western blot
Loading amount
50 µg
Gel Running Conditions
Non-reduced Denaturing (8%)
Sample
Mouse Cell lysate - whole cell (Primary hepatoctyes)
Specification
Primary hepatoctyes
Blocking step
I-Block(Applied biosystems) as blocking agent for 16 hour(s) and 0 minute(s) · Concentration: 2µg/mL · Temperature: 4°C
Read More

Mr. Shengyun Ma

Verified customer

投稿 Aug 09 2013

Question

I would like clarification on the predicted band size. I have read the following: "Question: don't these two statements from the datasheet contradict each other: 1. This antibody detects a band at 130kDa corresponding to Rad 21 and also a band of unknown origin at 75kDa. 2. WB: 1/500 - 1/5000. Predicted molecular weight: 72 kDa. unless 75kda is the unprocessed precursor and the protein is phosphorylated? thank you. Answer Thank you for your enquiry. I have been in contact with the originator of this product, and the band at 130 kDa is the Rad 21 band, and the band at 75 is unknown. I do not know exactly why Rad21 runs a larger band than is predicted. I hope this information helps, please do not hesitate to contact us if you need any more advice or information." On the datasheet it is implied 75kDa is the predicted weight. Does the datasheet need updating to 130kDa? Can you clarify the expected band size?When I run WB I get strong band at 130kDa.

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Answer



Based on Swiss-prot, the predicted molecular weight for this protein is indeed 75 kDa as mentioned on our datasheet. However, a band at ˜120-130 kDa has been previously observed: Mol Cell Biol. 2002 Dec;22(23):8267-77 PMID: 12417729. The higher molecular weight band seen is also consistent with the banding pattern observed for other commercially available antibodies to Rad21.

Some suggestions for the presence of these two bands is that the molecular weight of rad21 is ˜75kDa and the molecular weight of phosphorylated rad 21 is ˜120 kDa. Other data suggests that ab992 recognises endogenous Rad21 at 130 kDa in HeLa cell lysates consistent with the findings by Hoque and Ishikawa JBC, 276, 5059-5067, 2001 and that the prominent band seen at 75 kDa has not been characterised but is presumed to be a fragment of Rad21.

I am sorry that I do not have a more definitive answer for your but it would seem that the exact identity of these the bands has still not been confirmed.

With regards to the above Question and Answer published on our website, I have removed this as it was confusing and incorrect so thank you for bringing this to our attention.

Read More

1-10 of 27 Abreviews or Q&A

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