12-9865-42

antibody from Invitrogen Antibodies

Targeting: H2AX H2AFX

Flow cytometry (Supportive data in Antibodypedia)

Other assay (Supportive data in Antibodypedia)

Antibody Data

Product number

12-9865-42

Provider

Invitrogen Antibodies

Product name

Phospho-Histone H2A.X (Ser139) Monoclonal Antibody (CR55T33), PE, eBioscience™

Antibody type

Monoclonal

Antigen

Other

Description

Description: The CR55T33 monoclonal antibody recognizes phosphorylated serine 139 of human and mouse H2AX. H2AX is a member of the H2A histone family that complex with DNA and other histones to form the repeating nucleosome units characteristic of eukaryotic chromatin. Nucleosomes consist of approximately 147 base pairs of DNA wrapped around an octamer of histones composed of two each of the four histone proteins: H2A, H2B, H3 and H4. After induction of DNA damage such as double-strand breaks by irradiation, genotoxic stresses, replication errors or gene recombination, PI3K-like kinases (e.g., ataxia telangiectasia mutated (ATM), ataxia telangiectasia Rad-3-related (ATR), and DNA-dependent protein kinase (DNA-PK) are activated to phosphorylate serine 139 in H2AX. This early phosphorylation event plays a critical role in recruiting proteins involved in DNA repair. The monoclonal antibody CR55T33 recognizes a single band of approximately 15 kDa on reduced cell lysates from Jurkat cells stimulated with etoposide. Applications Reported: This CR55T33 antibody has been reported for use in intracellular staining followed by flow cytometric analysis. Applications Tested: This CR55T33 antibody has been pre-titrated and tested by intracellular staining and flow cytometric analysis of stimulated normal human peripheral blood cells using the Foxp3/Transcription Factor Staining Buffer Set (Product # 00-5523-00) and protocol. Please refer to Best Protocols: Protocol B: One step protocol for (nuclear) intracellular proteins located under the Resources Tab online. This can be used at 5 µL (0.25 µg) per test. A test is defined as the amount (µg) of antibody that will stain a cell sample in a final volume of 100 µL. Cell number should be determined empirically but can range from 10^5 to 10^8 cells/test. Excitation: 488-561 nm; Emission: 578 nm; Laser: Blue Laser, Green Laser, Yellow-Green Laser. Filtration: 0.2 µm post-manufacturing filtered.

Reactivity

Human, Mouse

Host

Mouse

Conjugate

Yellow dye

Isotype

IgG

Antibody clone number

CR55T33

Vial size

100 Tests

Concentration

5 µL/Test

Storage

4° C, store in dark, DO NOT FREEZE!

References

Submitted references

Olaparib-Resistant BRCA2(MUT) Ovarian Cancer Cells with Restored BRCA2 Abrogate Olaparib-Induced DNA Damage and G2/M Arrest Controlled by the ATR/CHK1 Pathway for Survival.

Biegała Ł, Gajek A, Marczak A, Rogalska A
Cells 2023 Mar 29;12(7)

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Adipocyte-mediated epigenomic instability in human T-ALL cells is cytotoxic and phenocopied by epigenetic-modifying drugs.

Lee M, Geitgey DK, Hamilton JAG, Boss JM, Scharer CD, Spangle JM, Haynes KA, Henry CJ
Frontiers in cell and developmental biology 2022;10:909557

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Microevolution of CG23-I Hypervirulent Klebsiella pneumoniae during Recurrent Infections in a Single Patient.

Wang YC, Lu MC, Li YT, Tang HL, Hsiao PY, Chen BH, Teng RH, Chiou CS, Lai YC
Microbiology spectrum 2022 Oct 26;10(5):e0207722

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The mitotic checkpoint is a targetable vulnerability of carboplatin-resistant triple negative breast cancers.

Moens S, Zhao P, Baietti MF, Marinelli O, Van Haver D, Impens F, Floris G, Marangoni E, Neven P, Annibali D, Sablina AA, Amant F
Scientific reports 2021 Feb 4;11(1):3176

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PRMT5 Modulates Splicing for Genome Integrity and Preserves Proteostasis of Hematopoietic Stem Cells.

Tan DQ, Li Y, Yang C, Li J, Tan SH, Chin DWL, Nakamura-Ishizu A, Yang H, Suda T
Cell reports 2019 Feb 26;26(9):2316-2328.e6

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ATR maintains chromosomal integrity during postnatal cerebellar neurogenesis and is required for medulloblastoma formation.

Lang PY, Nanjangud GJ, Sokolsky-Papkov M, Shaw C, Hwang D, Parker JS, Kabanov AV, Gershon TR
Development (Cambridge, England) 2016 Nov 1;143(21):4038-4052

Flow cytometry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Normal human peripheral blood cells were stimulated with Anti-CD3 and Anti-CD28 Functional Grade Purifieds (Product # 16-0037 and 16-0289) for 3 days. Cells were then unstimulated (left) or stimulated with etoposide (right) for 4 hours and then intracellularly stained with Anti-Human PARP1 (Cleaved) Alexa Fluor® 488 (Product # 53-6668-42) and Anti-Human/Mouse phospho-H2AX (S139) PE using the Foxp3/Transcription Factor Staining Buffer Set (Product # 00-5523) and protocol. Cells in the lymphocyte gate were used for analysis.

Conjugate

Yellow dye

Other assay

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

NULL

Conjugate

Yellow dye

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Proteome re-wiring of carboplatin-resistant 468-R cells protects from carboplatin-induced oxidative and genotoxic stress. ( A,B ) Hierarchical clustering of differentially expressed proteins (DE) ( A ) and Ingenuity Pathway Analysis ( B ) of the proteome alterations in MDA-MB-468 and 468-R cells treated with vehicle or 2 uM carboplatin for 5 days. Protein expression was analyzed by LC/MS-MS, and DE proteins were identified by ANOVA followed by Tukey's HSD test with an adjusted P value < 0.05. The top 10 significantly enriched pathways are shown. P value < 0.05 was used to determine significantly enriched pathways; the -log10 ( P value) was not shown if the pathway was not significantly enriched. N = 3. ( C,D ) ROS levels in MDA-MB-468 and 468-R cells treated with vehicle or 2 uM carboplatin for 5 days. Total ROS levels ( C ) were measured by the cellROX assay, and mitochondrial specific superoxide levels ( D ) were measured by the mitoSOX assay. Median flow of cytometric intensities were normalized to MDA-MB-468 vehicle condition. Data are shown as Mean +- SEM. P values were calculated by two-way ANOVA with Geisser-Greenhouse and Tukey's correction. N = 3. ( E ) NAD/NADH ratio as measured by NAD/NADH-Glo kit. Data are shown as Mean +- SEM. P values were calculated by two-way ANOVA with Geisser-Greenhouse and Tukey's correction. N = 3. ( F ) gamma-H2AX staining of MDA-MB-468 and 468-R cells treated with vehicle or 2 uM carboplatin for 5 days as analyzed by flow cytometry. P va

Conjugate

Yellow dye