Western blot analysis of extracts of HeLa, NCCIT and PYS2 cells using ATM (D2E2) Rabbit mAb.
Flow cytometric analysis of HeLa cells, untreated (left) or treated with camptothecin (1 μM, 2 hr; right), using Phospho-ATM (Ser1981) (D25E5) Rabbit mAb and Propidium Iodide (PI)/RNase Staining Solution #4087 to measure DNA content. Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
Immunoprecipitation of Rad51 from HeLa cell extracts using Rad51 (D4B10) Rabbit mAb (lane 2). Western blot detection was performed using the same antibody. Lane 1 is 10% input.
Western blot analysis of extracts from HT-29, HeLa, and MCF7 cells using BRCA1 (A8X9F) Rabbit mAb.
Western blot analysis of extracts from various cell lines using BRCA2 (D9S6V) Rabbit mAb (upper) or α-Actinin (D6F6) XP® Rabbit mAb #6487 (lower).
Immunoprecipitation of Rad54 from 293T cell extracts using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or Rad54 (D4W3Z) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot was performed using Rad54 (D4W3Z) Rabbit mAb.
Confocal immunofluorescent analysis of SK-MEL-28 (left) and GM07166 (right) cells using p95/NBS1 (D6J5I) Rabbit mAb (green). Actin filaments were labeled with DyLight™ 554 Phalloidin #13054.
Western blot analysis of extracts from Mv1Lu cells treated with UV or hydroxyurea (HU) for the indicated times, using Phospho-p95/NBS1 (Ser343) Antibody.
Western blot analysis of extracts from various cell lines using CtIP (D76F7) Rabbit mAb.
After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.
Western blot analysis of extracts from HCT 116 cells, untreated (-) or treated with neocarzinostatin (NCS 10 μM, 1 hr; +), using Phospho-ATM (Ser1981) (D25E5) Rabbit mAb (upper) and ATM (D2E2) Rabbit mAb #2873 (lower).
Western blot analysis of extracts from various cell lines using Rad51 (D4B10) Rabbit mAb.
Western blot analysis of DLD1 cells, either wild type (WT) or BRCA2 knockout (-/-), using BRCA2 (D9S6V) Rabbit mAb (upper) or α-Actinin (D6F6) XP® Rabbit mAb (lower). DLD1 (WT) and DLD1 (-/-) cells were kindly provided by Dr. Judit Jimenez, Yale University, New Haven, CT.
Western blot analysis of extracts from various cell lines using Rad54 (D4W3Z) Rabbit mAb.
Immunoprecipitation of p95/NBS1 from HeLa cell extracts using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or p95/NBS1 (D6J5I) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot was performed using p95/NBS1 (D6J5I) Rabbit mAb.
Western blot analysis of extracts from various cell lines using p95/NBS1 (D6J5I) Rabbit mAb (upper) and α-Actinin (D6F6) XP® Rabbit mAb #6487 (lower).
|ATM (D2E2) Rabbit mAb 2873||20 µl||
||H M||350||Rabbit IgG|
|Phospho-ATM (Ser1981) (D25E5) Rabbit mAb 13050||20 µl||
|Rad51 (D4B10) Rabbit mAb 8875||20 µl||
||H M R Mk||37||Rabbit IgG|
|BRCA1 (A8X9F) Rabbit mAb 14823||20 µl||
|BRCA2 (D9S6V) Rabbit mAb 10741||20 µl||
|Rad54 (D4W3Z) Rabbit mAb 15016||20 µl||
|p95/NBS1 (D6J5I) Rabbit mAb 14956||20 µl||
||H M R||95||Rabbit IgG|
|Phospho-p95/NBS1 (Ser343) Antibody 3001||20 µl||
||H M Mi||95||Rabbit|
|CtIP (D76F7) Rabbit mAb 9201||20 µl||
||H Mk||110||Rabbit IgG|
|Anti-rabbit IgG, HRP-linked Antibody 7074||100 µl||
The Homologous Recombination (HR) DNA Repair Antibody Sampler Kit provides an economical means of detecting proteins involved in HR DNA repair. The kit includes enough antibodies to perform two western blot experiments with each primary antibody.
Each antibody in the Homologous Recombination (HR) DNA Repair Antibody Sampler Kit detects endogenous levels of its target protein. Phospho-ATM (Ser1981) (D25E5) Rabbit mAb recognizes endogenous levels of ATM protein only when phosphorylated at Ser1981. Phospho-p95/NBS1 (Ser343) Antibody detects endogenous levels of p95/NBS1 protein only when phosphorylated at Ser343.
Monoclonal antibodies are produced by immunizing animals with recombinant human ATM, Rad51, BRCA1 and BRCA2, or synthetic peptides corresponding to residues surrounding Gly246 of human Rad54, Ala740 of human p95/NBS1, or residues near the carboxy terminus of human CtIP protein. Phosphorylation-specific monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ser1981 of human ATM. Phosphorylation-specific polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to Ser343 of human p95/NBS1 protein. Polyclonal antibodies are purified by peptide affinity chromatography.
DNA double-strand breaks (DSBs) are potentially hazardous lesions that can be induced by ionizing radiation (IR), radiomimetic chemicals, or DNA replication inhibitors. Cells recognize and repair DSBs via two distinct but partly overlapping signaling pathways, non-homologous end joining (NHEJ) and homologous recombination (HR). DSBs that arise during S or G2 phase are repaired via HR, using the replicated sister chromatid as a repair template (1). Activation of ATM by autophosphorylation on Ser1981 occurs in response to exposed DNA DSBs. ATM regulates various responses to DNA damage, including regulation of HR factors (2). Rad51 recombinase polymerizes and forms a filament along single-stranded DNA, mediating HR with the help of auxiliary proteins, including Rad54 and BRCA2 (3). BRCA2 has been shown to be required for localization of Rad51 to sites of DSBs, and cells lacking BRCA1 and BRCA2 cannot repair DSBs through HR (4). NBS1 is critical for HR, and requires CDK-dependent association with CtIP and subsequent phosphorylation by ATM at Ser278 and Ser343 (5-6).
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