Overview

  • Product nameAnti-Rad50 antibody [13B3/2C6]
    See all Rad50 primary antibodies
  • Description
    Mouse monoclonal [13B3/2C6] to Rad50
  • Tested applicationsSuitable for: Flow Cyt, IHC-P, ICC/IF, WB, IPmore details
  • Species reactivity
    Reacts with: Mouse, Human
  • Immunogen

    Fusion protein containing the complete coding region (amino acids 1-425) of RAD50 expressed in E.coli.

  • Positive control
    • Raji cell lysate HEK293 cell lysate
  • General notesClone 2C6 recognizes Rad50, a 153kDa protein involved in DNA double strand break repair. Rad50 is associated with Mre11 and p95 (Nibrin) to form a multiprotein complex involved in the double strand break repair process.

Properties

Applications

Our Abpromise guarantee covers the use of ab89 in the following tested applications.

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

Application Abreviews Notes
Flow Cyt Use 1µg for 106 cells.
IHC-P 1/400.
ICC/IF 1/200. See Abreview.
WB 1/1000. Detects a band of approximately 150 kDa (predicted molecular weight: 146 kDa).
IP Use at an assay dependent concentration.

Target

  • FunctionComponent of the MRN complex, which plays a central role in double-strand break (DSB) repair, DNA recombination, maintenance of telomere integrity and meiosis. The complex possesses single-strand endonuclease activity and double-strand-specific 3'-5' exonuclease activity, which are provided by MRE11A. RAD50 may be required to bind DNA ends and hold them in close proximity. This could facilitate searches for short or long regions of sequence homology in the recombining DNA templates, and may also stimulate the activity of DNA ligases and/or restrict the nuclease activity of MRE11A to prevent nucleolytic degradation past a given point. The complex may also be required for DNA damage signaling via activation of the ATM kinase. In telomeres the MRN complex may modulate t-loop formation.
  • Tissue specificityExpressed at very low level in most tissues, except in testis where it is expressed at higher level. Expressed in fibroblasts.
  • Involvement in diseaseDefects in RAD50 are the cause of Nijmegen breakage syndrome-like disorder (NBSLD) [MIM:613078]; also called NBS-like disorder or RAD50 deficiency. NBSLD is a disorder similar to Nijmegen breakage syndrome and characterized by chromosomal instability, radiation sensitivity, microcephaly, growth retardation, short stature and bird-like face. Immunodeficiency is absent.
  • Sequence similaritiesBelongs to the SMC family. RAD50 subfamily.
    Contains 1 zinc-hook domain.
  • DomainThe zinc-hook, which separates the large intramolecular coiled coil regions, contains 2 Cys residues that coordinate one molecule of zinc with the help of the 2 Cys residues of the zinc-hook of another RAD50 molecule, thereby forming a V-shaped homodimer. The two heads of the homodimer, which constitute the ATP-binding domain, interact with the MRE11A homodimer.
  • Post-translational
    modifications
    Phosphorylated upon DNA damage, probably by ATM or ATR.
  • Cellular localizationNucleus. Chromosome > telomere. Localizes to discrete nuclear foci after treatment with genotoxic agents.
  • Information by UniProt
  • Database links
  • Alternative names
    • DNA repair protein RAD50 antibody
    • hRAD50 antibody
    • hsRAD50 antibody
    • Mrell antibody
    • NBSLD antibody
    • Rad 50 antibody
    • RAD50 2 antibody
    • rad50 antibody
    • RAD50 homolog (S cerevisiae) antibody
    • RAD50 homolog antibody
    • RAD50 PEN antibody
    • RAD50, S. cerevisiae, homolog of antibody
    • RAD50_HUMAN antibody
    • RAD502 antibody
    • Rad50l antibody
    • Truncated RAD50 protein antibody
    see all

Anti-Rad50 antibody [13B3/2C6] images



  • Predicted band size : 146 kDa


    This picture was kindly supplied as part of the reviews for ab214 and for ab89 submitted by Anya Polischouk.

    The bands represent nuclear (N) and cytoplasmic (C) extract from human lung cancer cells (U1810).

  • Anti-Rad50 antibody [13B3/2C6] (ab89) at 1/1000 dilution + 50 ug Human HEK293 total cell lysate

    Secondary
    HRP conjugated Donkey Anti-Mouse IgG
    Developed using the ECL technique

    Performed under non-reducing conditions.

    Predicted band size : 146 kDa
    Observed band size : 160 kDa (why is the actual band size different from the predicted?)
    Additional bands at : 300 kDa (possible non-specific binding).

    Exposure time : 30 seconds

    See Abreview

  • ab89 staining Rad50 in HPV transfected Hela cells by Immunocytochemistry/ Immunofluorescence. Cells were washed twice with phosphate-buffered saline (PBS), permeabilized with 0.5% Triton X-100 in CSK buffer (10 mM Hepes-KOH, pH 7.4; 300 mM sucrose; 100 mM NaCl; 3 mM KCl) for 2 min on ice, and fixed with 4% paraformaldehyde. Fixed cells were treated with 0.5% Triton X-100 in PBS, followed by 3 PBS washes for 5 min each at RT. After blocking with 3% bovine serum albumin (BSA) in PBS at RT for 30 min, the cells were incubated with primary antibody in antibody-binding solution (3% BSA in PBS) at RT for 30 min. An Alexa Fluor® 488 conjugated anti mouse secondary was used.

  • ab89 (1µg/ml) staining RAD50 in human placenta, using an automated system (DAKO Autostainer Plus). Using this protocol there is strong nuclear staining.
    Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer EDTA pH 9.0 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.
  • Overlay histogram showing HeLa cells stained with ab89 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab89, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

References for Anti-Rad50 antibody [13B3/2C6] (ab89)

This product has been referenced in:
  • Clynes D  et al. ATRX dysfunction induces replication defects in primary mouse cells. PLoS One 9:e92915 (2014). IP, IF ; Mouse . Read more (PubMed: 24651726) »
  • Koppensteiner R  et al. Effect of MRE11 Loss on PARP-Inhibitor Sensitivity in Endometrial Cancer In Vitro. PLoS One 9:e100041 (2014). IHC-P ; Human . Read more (PubMed: 24927325) »

See all 16 Publications for this product

Product Wall

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Sample Human Cell lysate - whole cell (SW480 cells)
Loading amount 25 µg
Specification SW480 cells
Gel Running Conditions Reduced Denaturing (4-12%)
Username

Abcam user community

Verified customer

Submitted Feb 05 2013

Thank you for your enquiry. I was looking at the Swiss Prot entry for the protein that this antibody is specific too, and the tissue specificity is listed as: Expressed at very low level in most tissues, except in testis where it is expressed at hig...

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Sample Human Cell lysate - whole cell (lung and foreskin fibroblast cells)
Specification lung and foreskin fibroblast cells
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5%
Username

Abcam user community

Verified customer

Submitted Mar 02 2006

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Sample Human Cell lysate - whole cell (HEK293)
Specification HEK293
Blocking step Milk as blocking agent for 30 minute(s) · Concentration: 5%
Username

Dr. Philippe Szankasi

Verified customer

Submitted Feb 24 2006

Thank you for inquiring about our RAD50 monoclonal antibody (clone 13B3). The exact epitope that this antibody recognizes has not been determined. However, it is quite possible that the epitope is near or around the amino terminus of RAD50 as the immun...

Read More

Dear Carmela, Have Genetex replied to you on this one yet? Abcam

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