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Ab76420

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Ab76420 is a laboratory equipment product. It is designed for a specific function in a research or diagnostic laboratory setting. No further details about the core function or intended use of this product are provided.

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Lab products found in correlation

Ab15090, by Abcam (2 mentions) Ab97672, by Abcam (2 mentions) Ab133534, by Abcam (2 mentions) Ab15093, by Abcam (1 mentions) Ab150084, by Abcam (1 mentions) Ab150120, by Abcam (1 mentions) Ab150113, by Abcam (1 mentions) Sc 22768, by Merck Group (1 mentions) Vectashield antifade mounting medium with dapi, by Vector Laboratories (1 mentions) Ab150077, by Abcam (1 mentions) Protease and phosphatase inhibitor cocktail, by Beyotime (1 mentions) Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions) Horseradish peroxidase hrp, by Cell Signaling Technology (1 mentions) A5441, by Merck Group (1 mentions) Tween 20, by Beyotime (1 mentions) Pvdf membrane, by Merck Group (1 mentions) Chemidoc touch, by Bio-Rad (1 mentions) Skim milk, by BD (1 mentions) Radioimmunoprecipitation assay (ripa), by Beyotime (1 mentions) A11070, by Thermo Fisher Scientific (1 mentions)

9 protocols using ab76420

1

Immunofluorescence Analysis of Meiotic Chromosomes

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Spermatocyte and oocyte chromosome spreading was prepared as previously described (50 (link), 51 (link)). Structurally preserved spermatocytes were prepared as described previously (52 (link)).
Primary antibodies used for immunofluorescence were as follows: rabbit anti-SYCP3 (1:500 dilution; Abcam #ab15093), mouse anti-SYCP3 (1:500 dilution; Abcam #ab97672), rabbit anti-SYCP1 (C-terminal) (1:2000 dilution; Abcam #ab15090), rabbit anti-SYCP1 (N-terminal) (1:2000 dilution; Abclonal #A12139), rabbit anti-RPA2 (1:200 dilution; Abcam #ab76420), rabbit anti-RAD51 (1:200 dilution; Thermo Fisher Scientific #PA5-27195), rabbit anti-DMC1 (1:100 dilution; Santa Cruz Biotechnology #sc-22768), mouse anti–phospho-histone H2AX (pSer139) (1:300 dilution; Millipore #05-636), mouse anti-MLH1 (1:50 dilution; BD Biosciences #550838), rabbit anti-TEX12 (1:1000 dilution; Proteintech #17068-1-AP), mouse anti-TRF1 (1:1000 dilution; homemade), and rabbit anti-BRCA1 (1:500 dilution; a gift from L.-Y. Lu, Zhejiang University). Primary antibodies were detected with Alexa Fluor 488– or 594–conjugated secondary antibodies (1:500 dilution; Abcam #ab150084, #ab150077, #ab150113, and #ab150120) for 1 hour at room temperature. The slides were washed several times with PBS and mounted using VECTASHIELD antifade mounting medium with DAPI (Vector Laboratories, #H-1200).
Li M., Huang T., Li M.J., Zhang C.X., Yu X.C., Yin Y.Y., Liu C., Wang X., Feng H.W., Zhang T., Liu M.F., Han C.S., Lu G., Li W., Ma J.L., Chen Z.J., Liu H.B, & Liu K. (2019). The histone modification reader ZCWPW1 is required for meiosis prophase I in male but not in female mice. Science Advances, 5(8), eaax1101.
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2

Western Blot Analysis of Cellular Proteins

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With a phosphatase and protease inhibitor cocktail (Beyotime, China), cells were lysed and sonicated in RIPA (Beyotime, China) lysis solution. Proteins were separated by SDS‒PAGE and then transferred to a PVDF membrane (Merck Millipore, USA). Following 1 h at room temperature in a buffer containing 5% skim milk (BD Biosciences, USA) and 0.1% Tween 20 (Beyotime, China), the membranes were blocked. The membrane was then treated with the primary antibodies specified overnight at 4 °C, followed by incubation with goat anti-mouse or anti-rabbit secondary antibodies conjugated with horseradish peroxidase (HRP) (Cell Signaling Technology, USA) for 1 h. PierceTM ECL western blotting substrate (Thermo Fisher, USA) and Bio-Rad ChemiDoc Touch were used to detect and analyze the proteins. Antibodies against the following targets were used in this study: phospho-mTOR, mTOR, LC3B, P62, cleaved caspase 3, γ-H2A (5536, 2983, 3868, 23214, 9661, 2577, Cell Signaling Technology, USA), phospho-RPA2 (381220, Zen BioScience, China), actin (A5441, Sigma‒Aldrich, USA), RPA2 (ab76420, Abcam, USA), and Ki-67 (14-5698-82, Thermo Fisher, USA).
Feng Y., Jiang Y., Liu J., Liu J., Shi M., Chen J., Zhang J., Tian Y., Yang X, & Liu H. (2023). Targeting RPA promotes autophagic flux and the antitumor response to radiation in nasopharyngeal carcinoma. Journal of Translational Medicine, 21, 738.
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3

Immunofluorescence Analysis of Meiotic Chromosome Proteins

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Ovaries were dissected from freshly euthanized animals and processed for surface spreading of oocyte chromosomes as described (Sun and Cohen, 2013 (link)). Immunofluorescence staining was performed using the following primary antibodies with incubation overnight at room temperature: mouse anti-SYCP3 (sc-74568 Santa Cruz, 1:200 dilution), rabbit anti-SYCP3 (sc-33195 Santa Cruz, 1:300), guinea pig anti-SYCP2 (a gift from Christer Höög, 1:300) (Kouznetsova et al., 2005 (link)), rabbit monoclonal anti-RPA32 (ab76420 Abcam, 1:100), rabbit anti-REC8 (a gift from Scott Keeney, 1:300), mouse monoclonal anti-γH2AX (05-636 Millipore, 1:500), rabbit polyclonal anti-γH2AX (A300-081A Bethyl laboratories, 1:300), goat polyclonal anti-Caspase3 (L-18 Sc-1225, 1:100), rabbit anti-HORMAD1 (a gift from Attila Toth, 1:250)(Wojtasz et al., 2009 (link)). Slides were subsequently incubated with the following goat secondary antibodies for 1 h at 37 °C: anti-rabbit 488 (A11070 Molecular Probes, 1:1000 dilution), anti-rabbit 568 (A11036 Molecular Probes, 1:2000), anti-mouse 555 (A21425 Molecular Probes, 1:1000), anti-mouse 594 (A11020 Molecular Probes, 1:1000), anti-mouse 488 (A11029 Molecular Probes, 1: 1000), or anti-guinea pig fluorescein isothiocyanate (106-096-006 FITC, Jackson Labs, 1:200). Coverslips were mounted with ProLong Gold antifade reagent (Molecular Probes).
Qiao H., Prasada Rao H.B., Yun Y., Sandhu S., Fong J.H., Sapre M., Nguyen M., Tham A., Van B.W., Chng T.Y., Lee A, & Hunter N. (2018). Impeding DNA Break Repair Enables Oocyte Quality Control. Molecular cell, 72(2), 211-221.e3.
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4

Antibody Panel for DNA Damage Response

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The following antibodies were used: anti-ZGRF1 (LS-C168135,1:1000 for WB,LSBio), anti-BRCA1 (D-9, dilution:1:100 for IF, Santa Cruz) and (C-20,1:200 for WB and IP, Santa Cruz), anti-EXO1 (ab95012,1:1000 forWB, Abcam), anti-BLM (ab2179, 1:1000 for WB, Abcam), anti-CHK1 pS317 (2344 S, 1:500 for WB, Cell Signaling), anti-CHK1 (sc-7898, 1:1000 for WB, Santa Cruz), anti-CHK2 pT68 (GTX61178, 1:1000 for WB, GeneTex), anti-CHK2 (2662,1:1000 for WB, Cell Signaling Technology), anti-RPA2 pS4/S8 (A300-245A, 1:1000 for WB, Bethyl Laboratories), anti-RPA2 (ab76420; 1:500 for IF; Abcam), anti-γH2AX (05-636; 1:1000 for WB and 1:500 for IF; Millipore)
Yan S., Song M., Ping J., Lai S.T., Cao X.Y., Bai C.J., Xie D.F., Guan H., Gao S.S, & Zhou P.K. (2021). ZGRF1 promotes end resection of DNA homologous recombination via forming complex with BRCA1/EXO1. Cell Death Discovery, 7, 260.
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5

Chromosome Spreading and Immunofluorescence Analysis

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Spermatocyte and oocyte chromosome spreading was performed as previously described (24 (link)). Produced antibody used for western blotting: Mouse anti-SPIDR polyclonal antibody was prepared against mouse SPIDR protein by Dai-an Biological Technology Incorporation (Wuhan, China). Primary antibodies used for immunofluorescence were as following: rabbit anti-SYCP1 (1:500 dilution; Abcam #ab15090), mouse anti-SYCP3 (1:500 dilution; Abcam #ab97672), rabbit anti-RPA2 (1:200 dilution; Abcam #ab76420), rabbit anti-RAD51 (1:200 dilution; Thermo Fisher Scientific #PA5-27195), rabbit anti-DMC1 (1:200 dilution; homemade); mouse anti-γH2AX (pSer139) (1:1000 dilution; Millipore #05–636), mouse anti-MLH1 (1:100 dilution; BD Biosciences #550838), rabbit anti-BLM (1:200 dilution; Invitrogen #PA5-27384), rabbit anti-MSH4(1:200 dilution; Abcam #ab58666); rat anti-HEI10 (1:100 dilution; homemade); Rabbit anti-MSY2(1:200 dilution; Abcam #ab33164). Primary antibodies were detected with Alexa Fluor 488- or 594-conjugated secondary antibodies (1:500 dilution; Abcam #ab150080, #ab150077, #ab150113 and #ab150116) for 1 h at room temperature. The slides were mounted using Mounting Medium with DAPI (Abcam #ab104139).
Huang T., Wu X., Wang S., Bao Z., Wan Y., Wang Z., Li M., Yu X., Lv Y., Liu Z., Chen X., Chan W.Y., Gao F., Lu G., Chen Z.J, & Liu H. (2023). SPIDR is required for homologous recombination during mammalian meiosis. Nucleic Acids Research, 51(8), 3855-3868.
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6

Laser Microirradiation and DNA Damage Response

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Laser microirradiation was performed as described previously (Eid et al., 2010) . Briefly, 10 mM BrdU was added to cells 24h prior to irradiation. Microirradiation was performed using a MMI CELLCUT system containing a UV A laser of 355 nm (Molecular Machines and Industries, Zurich, Switzerland). The laser intensity was set to 50% energy output and each cells was exposed to the laser beam for < 300ms. After a release of 2h cells were fixed in 4% formaldehyde (w/v) in PBS for 15 min and permeabilized with Triton X-100 (0.5% in PBS) for 5 min at room temperature. Subsequently, cells were blocked for 1h in 3% FCS (w/v) in PBS and stained with primary antibodies 53BP1 (abcam, ab21083, RRID:AB_722496, rabbit, 1:500) and RPA (EPR2877Y, abcam, ab76420, RRI-D:AB_1524336, rat, 1:100). After staining with appropriate secondary antibodies Alexa Fluor-488 rabbit and Alexa Fluor-594 rat (1:1000) (Life Technologies), coverslips were mounted with Vectashield (Vector Laboratories) containing DAPI and sealed. Images were acquired on a Leica DMI6000. RPA co-localization with 53BP1 was analyzed by fluorescence microscopy.
Francica P., Mutlu M., Blomen V.A., Oliveira C., Nowicka Z., Trenner A., Gerhards N.M., Bouwman P., Stickel E., Hekkelman M.L., Lingg L., Klebic I., van de Ven M., de Korte-Grimmerink R., Howald D., Jonkers J., Sartori A.A., Fendler W., Chapman J.R., Brummelkamp T, & Rottenberg S. (2020). Functional Radiogenetic Profiling Implicates ERCC6L2 in Non-homologous End Joining. Cell reports, 32(8).
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7

Affinity Capture of RPA from HeLa Cells

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Peptides (EV-34, pEV-34 and ppEV-34) were covalently coupled to epoxy beads (Dynabeads M-270 Epoxy, Thermo Fisher) as described by the producer. Coupled beads (15 μl) were added to HeLa whole cell extract (WCE, 1 mg protein) and incubated for 30 min before washing in PBS and elution in LDS loading buffer. Input and affinity captured RPA were quantified by western analysis using monoclonal rabbit anti-RPA2 [EPR2877Y] (ab76420) primary antibody (1:1000, Abcam) and swine anti-rabbit HRP (1:5000, Dako) as secondary antibody.
Kavli B., Iveland T.S., Buchinger E., Hagen L., Liabakk N.B., Aas P.A., Obermann T.S., Aachmann F.L, & Slupphaug G. (2021). RPA2 winged-helix domain facilitates UNG-mediated removal of uracil from ssDNA; implications for repair of mutagenic uracil at the replication fork. Nucleic Acids Research, 49(7), 3948-3966.
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8

Antibody Validation for Cellular Studies

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Anti-FAM35A rabbit polyclonal antibodies (1:500 for Western-blotting [WB]) are purchased from Abcam (ab136079), anti-C20ORF196 rabbit polyclonal antibodies (1:500 for WB) from Sigma-Aldrich (hpa040749), anti-REV7 rabbit polyclonal antibodies (1:2000 for WB) from BD Biosciences (612266), anti-RAD51 rabbit polyclonal antibodies (1:200 for immunofluorescence [IF]) from Abcam (ab133534), anti-POLD3 rabbit polyclonal antibodies (1:2000 for WB) from Abnova (H00010714-M01), anti-GFP rabbit polyclonal antibodies (1:1000 for WB) from Proteintech Group (66002-1-Ig), anti-β-actin mouse monoclonal antibody (1:5000 for WB) from MBL (M177-3), anti-FLAG M2 monoclonal antibody (1:5000 for WB) from Sigma-Aldrich (F3165), anti-RPA32 rabbit monoclonal antibody (1:500 for IF) from Abcam (ab76420) and anti-pRPA32 (S4/S8) rabbit polyclonal antibodies (1:500 for IF) from Bethyl (A300-245A).
Gao S., Feng S., Ning S., Liu J., Zhao H., Xu Y., Shang J., Li K., Li Q., Guo R, & Xu D. (2018). An OB-fold complex controls the repair pathways for DNA double-strand breaks. Nature Communications, 9, 3925.
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9

Comprehensive PARP Inhibitor and DNA Damage Assessment

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The PARPi, niraparib (MK4827), rucaparib (AG014699), veliparib (ABT-888), olaparib (AZD2281), talazoparib (BMN-673); the topoisomerase II inhibitor, etoposide (HY-13629); and the alkylating agent, temozolomide (HY-17364), doxorubicin (HY-15142A), ifosfamide (HY-17419) and dacarbazine (HY-B0078) were purchased from MedChemExpress (New Jersey, USA). The anti-PARP1 antibody (ab32071), the anti-poly (ADP-Ribose) polymer [10H] (ab14459), the anti-Rad51 antibody (ab133534), the anti-RPA32 antibody (ab76420), the anti-Ki67 antibody (ab15580), and the goat anti-rabbit IgG H&L (Alexa Fluor® 488) (ab150077) were purchased from Abcam (Cambridge, UK). Anti-phospho-H2AX (S139) was purchased from Affymetrix eBioscience (Massachusetts, USA). Anti-β-actin (13E5) and anti-GAPDH (14C10) were purchased from Cell Signaling Technology (Massachusetts, USA). Drugs and antibodies were used according to the associated protocols.
Li H., Tu J., Zhao Z., Chen L., Qu Y., Li H., Yao H., Wang X., Lee D.F., Shen J., Wen L., Huang G, & Xie X. (2020). Molecular signatures of BRCAness analysis identifies PARP inhibitor Niraparib as a novel targeted therapeutic strategy for soft tissue Sarcomas. Theranostics, 10(21), 9477-9494.
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