Anti rpa32

Manufactured by GeneTex

Anti-RPA32 is a primary antibody specific for the RPA32 (RPA2) subunit of the eukaryotic Replication Protein A (RPA) complex. RPA is a single-stranded DNA-binding protein complex that plays a critical role in DNA replication, repair, and recombination.

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Lab products found in correlation

Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions) Etoposide, by Merck Group (2 mentions) Diamidino 2 phenylindole dapi, by Thermo Fisher Scientific (2 mentions) Anti 53bp1, by Cell Signaling Technology (2 mentions) Axio imager z1, by Zeiss (2 mentions) 6 well tissue culture treated plates, by Greiner (2 mentions) Anti flag m2, by Merck Group (2 mentions) Anti γh2ax, by GeneTex (1 mentions) Anti vinculin, by Abcam (1 mentions) Las 3000 imager, by Fujifilm (1 mentions) β actin antibody, by Merck Group (1 mentions)

3 protocols using anti rpa32

Quantifying DNA Damage Response Markers

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Cells were plated in 6-well tissue culture-treated plates (Greiner) at 1.75 × 10⁶ cells/well and allowed to rest overnight. Cells were then infected with rAAV 2.5 at equal titers (1.4 × 10⁸ copies/well) or 50 μM etoposide (Sigma) for 20 h. For the EdU-IF experiments, EdU was added to the cells for 20 min. Cells were then permeabilized with 0.5% Triton X-100 in PBS at 4°C for 5 min and fixed in 4% PFA for 20 min. Samples were then washed in 1× PBS and incubated with blocking buffer (3% BSA, 0.05% Tween 20, and 0.04 NaN₃ in PBS) for 30 min. Cells were probed with appropriate primary antibodies (anti-FLAG M2 [Sigma-Aldrich], anti-γH2AX, anti-RPA32 [GeneTex], or anti-53BP1 [Cell Signaling]) and then washed in PBST (0.05% Tween 20 in PBS) and probed with Alexa Fluor-conjugated secondary antibodies (Life Technologies). Nuclei were stained with diamidino-2-phenylindole (DAPI; Life Technologies). Secondary staining for EdU was added as the last step and stained twice to ensure signal. Images were acquired on the Zeiss Axio Imager Z1, and mean fluorescence intensity (MFI) was analyzed using ImageJ.

Li D., Lopez A., Sandoval C., Nichols Doyle R, & Fregoso O.I. (2020). HIV Vpr Modulates the Host DNA Damage Response at Two Independent Steps to Damage DNA and Repress Double-Strand DNA Break Repair. mBio, 11(4), e00940-20.

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Western Blotting Analysis of Cell Extracts

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The analysis of cell extracts by western blotting was performed as described previously [9 (link)]. For ATR analysis, 6% SDS-PAGE gels were used. Antibodies against phospho-CHK1 Ser345 and phospho-PP1α Thr320 were purchased from Cell Signaling; anti-ATR, anti- CHK1, anti-CdC25A, anti-PP1α and anti-PP2A antibodies were from Santa-Cruz Biotechnology; anti-RPA32 was from Genetex and phospho-RPA32 Ser33 was from Bethyl Laboratories. The anti-Vinculin and phospho-PP2A Tyr307 antibodies were purchased from Abcam. The β-actin antibody was purchased from Sigma-Aldrich and the anti-adaptin antibody from BD Biosciences. The anti-Spi1 antibody was described previously [9 (link)]. Specific peroxidase-conjugated secondary antibodies were used to detect protein expression using the LAS-3000 imager (Fujifilm). Images were cropped using Photoshop software (Adobe Systems France, Paris, France).

Rimmelé P., Esposito M., Delestré L., Guervilly J.H., Ridinger-Saison M., Despras E., Moreau-Gachelin F., Rosselli F, & Guillouf C. (2017). The Spi1/PU.1 transcription factor accelerates replication fork progression by increasing PP1 phosphatase in leukemia. Oncotarget, 8(23), 37104-37114.

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Quantifying DNA Damage Responses

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Cells were plated in 6-well tissue culture treated plates (Greiner) at 1.75 x 10 6 cell/ well and allowed to rest overnight. Cells were then infected with rAAV 2.5 at equal titers (1.4 x 10 8 copies/ well) or 50uM etoposide (Sigma) for 20 hrs. For the EdU-IF experiments, EdU was added to the cells for 20 minutes. Cells were then permeabilized with 0.5% Triton X-100 in PBS at 4˚C for 5 min, fixed in 4% PFA for 20 min. Samples were then washed in 1X PBS and incubated with blocking buffer (3% BSA, 0.05% Tween-20, and 0.04 NaN3 in PBS) for 30 minutes. Cells were probed with appropriate primary antibodies (anti-FLAG M2 [Sigma-Aldrich], anti-gH2AX, anti-RPA32 [GeneTex], or anti-53BP1 [Cell Signaling]), then washed in PBST (0.05% Tween-20 in PBS), and probed with Alexa-Fluor conjugated secondary antibodies (Life Technologies). Nuclei were stained with diamidino-2-phenylindole (DAPI; Life Technologies). Secondary staining for EdU was added as the last step and stained twice to ensure signal. Images were acquired on the Zeiss Axioimager Z1 and mean fluorescence intensity (MFI) was analyzed using ImageJ.

Li D., Lopez A., Sandoval C., Doyle R.N., & Fregoso O.I. (2020). HIV Vpr modulates the host DNA damage response at two independent steps to damage DNA and repress double-strand DNA break repair.

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