United Kingdom (UK)
Recombinant monoclonal antibody to XRCC3. Manufactured using AbAb’s Recombinant Platform with variable regions (i.e. specificity) from the hybridoma 10F1/6.
UniProt Accession Number of Target Protein: O43542
Alternative Name(s) of Target: X-Ray Repair Cross Complementing 3; X-ray repair cross-complementing protein 3; DNA repair protein XRCC3; X-ray repair complementing defective repair in Chinese hamster cells 3; CMM6; 10F1
Immunogen: This antibody was raised by immunising mice with the denaturated recombinant human XRCC3-His6 protein which had been purified from 2.4 liters of E. coli FB810 recA− pLysS carrying pXRCC3-28c.
Specificity: This antibody is specific for XRCC3 and does not cross-react with Rad51B, Rad51C, Rad51D, Rad51, or XRCC2 in Western blot analysis. XRCC3 is a protein involved in the homologous recombination repair (HRR) pathway in chromosomal fragmentations and deletions.
Application Notes: WB: XRCC3 10F1/6 antibody was used to visualise XRCC3 on Western blots in multiple experiments (e.g. after previous gel filtration of infected pf9 cells; after immunoprecipitation with different antibodies and others) in a study on the RAD51 paralogs' complexes and their role in genetic recombination (Masson et al., 2001). WB: Sage et al. (2010) used this antibody to visualise XRCC3 protein on Western blots from lysates of HCT116 cells in the experiments demonstrating engagement of Rad51 proteins in mtDNA maintenance. ICC/IF: XRCC3 protein was immunostained with XRCC3 10F1/6 antibody in HEK293 cells (some of the cells had been subjected to the ionising radiation) in an experiment investigating the exact role of XRCC3 in homologous recombination (Forget et al., 2004).
Antibody first published in:
Masson et al. Complex formation by the human RAD51C and XRCC3 recombination repair proteins. Proc Natl Acad Sci U S A. 2001 Jul 17;98(15):8440-6. PMID:11459987
Note on publication:
This article describes the detailed generation of this anti-XRCC3 antibody as well as successfully applies it in Western analysis.
Immunofluorescence staining of HeLa cells using anti-XRCC3 antibody (Ab01310) 10F1/6. Immunofluorescence analysis of paraformaldehyde fixed HeLa cells permeabilized with 0.15% Triton and stained with the chimeric mouse IgG version of 10F1/6 (Ab01310-1.1) at 10 µg/ml followed by Alexa Fluor® 488 secondary antibody (2 µg/ml), showing nuclear staining. Actin filaments were stained with phalloidin (red) and the nuclear stain is DAPI (blue). Panels show from left-right, top-bottom Ab01310-1.1, DAPI, merged channels and an isotype control. The isotype control was stained with anti-unknown antibody (Ab00178-1.1) followed by Alexa Fluor® 488
Flow-cytometry using the anti-XRCC3 antibody 10F1/6 (Ab01310). HeLa cells were fixed using paraformaldehyde, permeabilised using 0.5% Triton and stained with anti-unknown IgG antibody (Ab00178-1.1; isotype control, black line) or the mouse IgG-chimeric version of 10F1/6 (Ab01310-1.1, blue line) at a dilution of 1:100 for 1h at RT. After washing, bound antibody was detected using a goat anti-mouse IgG AlexaFluor® 488 antibody at a dilution of 1:1000 and cells analyzed using a FACSCanto flow-cytometer.
Western Blot using anti-XRCC3 antibody 10F1/6 (Ab01310). nuclear lysates of HeLa(A) and HepG2(B) cells (35µg protein in RIPA buffer) was resolved on an SDS PAGE gel and blots probed with the chimeric mouse IgG version of 10F1/6 (Ab01310-1.1) at 2 µg/ml before detection using an anti-mouse secondary antibody. A primary incubation of 1h was used and protein was detected by chemiluminescence.
Flow cytometry using the Anti-XRCC3 antibody 10F1/6 (Ab01310). Paraformaldehyde fixed HeLa cells permeabilized with 0.5% Triton were stained with anti-unknown specificity antibody (Ab00178-1.1; isotype control, black line) or the mouse IgG1 version of 10F1/6 (Ab01310-1.1, blue line) at a dilution of 1:100 for 1h at RT. After washing, the bound antibody was detected using a goat anti-mouse IgG AlexaFluor® 488 antibody at a dilution of 1:1000 and cells analyzed using a FACSCanto flow-cytometer.