Rpa70

Manufactured by Merck Group

The RPA70 is a laboratory equipment product developed by Merck Group. It is a key component used in various scientific applications. The core function of the RPA70 is to perform specialized tasks in controlled research and analytical environments.

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Lab products found in correlation

γh2ax, by Cell Signaling Technology (2 mentions) Rad51, by Santa Cruz Biotechnology (2 mentions) Cyclin a, by Santa Cruz Biotechnology (2 mentions) P atm ser1981, by Santa Cruz Biotechnology (1 mentions) P nbs1 ser343, by Cell Signaling Technology (1 mentions) Fancd2, by Abcam (1 mentions) Gapdh, by Santa Cruz Biotechnology (1 mentions) Apotome fluorescence microscope, by Zeiss (1 mentions) Axiovision axiovs40 v 4, by Zeiss (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions) Hoechst, by Thermo Fisher Scientific (1 mentions) Vectashield mounting medium, by Vector Laboratories (1 mentions) Observer z1 microscope, by Zeiss (1 mentions)

3 protocols using rpa70

Immunoblotting Methodology and Antibody Validation

Cited 2 times

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Immunoblotting was performed as described [31] (link), [47] (link). Rabbit monoclonal anti-hMOB2 antibodies were produced in collaboration with Epitomics. The following antibodies were used in immunoblotting: ATM (Millipore, 07-1286, 1/1000), p-ATM Ser1981 (Santa Cruz, sc-47,739, 1/200), BRCA2 (Calbiochem, OP95, 1/500), p-BRCA2 Ser3291 [49] (link), FANCD2 (Abcam, ab108928, 1/500), RAD51 (Santa Cruz, sc-8349, 1/1000), p-RAD51 Ser14 [50] (link), RPA70 (Millipore, NA13, 1/100), RPA32/34 (Millipore, NA18, 1/1000), p-RPA32/34 Ser4/8 (Bethyl Labs, A300-245A, 1/1000), NBS1 (BD Biosciences, 611,870, 1/1000), p-NBS1 Ser343 (Cell Signalling, 3001, 1/500), p53 (Santa Cruz, sc-126, 1/1000), γH2AX (Cell Signalling, 9718, 1/500), HA (Cell Signalling, 3724, 1/1000) and GAPDH (Santa Cruz, sc-32,233, 1/1000). Polyclonal rat anti-tubulin (YL1/2) was produced in our laboratory. Densitometric analysis of immunoblots were performed using the ImageJ software (NIH).

Gundogdu R., Erdogan M.K., Ditsiou A., Spanswick V., Garcia-Gomez J.J., Hartley J.A., Esashi F., Hergovich A, & Gomez V. (2021). hMOB2 deficiency impairs homologous recombination-mediated DNA repair and sensitises cancer cells to PARP inhibitors. Cellular Signalling, 87, 110106.

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Immunofluorescence Staining of DNA Repair Proteins

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Cells were processed for immunofluorescence as described [31] (link), [51] (link). Briefly, cells cultured on glass coverslips were fixed in 3%-paraformaldehyde/2%-sucrose solution for 15 min at room temperature, permeabilized for 2 min with 0.5% (vol/vol) Triton X-100 in PBS, blocked (10% goat serum in PBS) and incubated with primary antibodies overnight at 4C. The next day, following incubation with secondary antibodies and DAPI (Sigma) or Hoechst (Invitrogen, H3570) for 2 h, coverslips were mounted using Vectashield mounting medium (Vector Lab). Primary and secondary antibodies were used in immunofluorescence as follows: RAD51 (Santa Cruz, sc-8349, 1/50) (Abcam, ab63801, 1/1000), RPA70 (Millipore, NA13, 1/100), γH2AX (Cell Signalling, 9718, 1/50), cyclin-A (Santa Cruz, sc-271,682, 1/100) and Mitosin/CENPF (Abcam, Ab5, 1/200), anti-rabbit FITC (Stratech – Jackson, 711-095-152, 1/100), anti-mouse FITC (Stratech – Jackson, 715-095-151, 1/100), anti-rabbit Texas Red (Stratech – Jackson, 711-075-152, 1/100) and anti-mouse Texas Red (Stratech – Jackson, 715-075-151, 1/100). Images were acquired with an ApoTome fluorescence microscope (Zeiss) with a 40× objective lens and processed with AxioVision AxioVS40 V4.8.1.0 (Zeiss) and Photoshop CS5 (Adobe Systems Inc.).

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Detecting DNA Damage Foci and Bridges

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RPA foci and anaphase bridges were detected in cells fixed with 4% paraformaldehyde and permeabilized with 0.2% TritonX-100. For RPA foci, cells were pre-extracted with 0.5% TritonX-100 before fixing. Antibodies used were RPA70 (Millipore), 53BP1 (Novus Biologicals), Cyclin A (Santa Cruz). The slides were mounted in Fluoromount-G 105 and is also made available for use under a CC0 license.
(which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC
The copyright holder for this preprint this version posted June 29, 2020. ; https://doi.org/10.1101/2020.06.29.177352 doi: bioRxiv preprint IR induces replication stress 22 (Southern Biotech). Images were acquired with a 63x oil immersion objective on a Zeiss Observer Z1 microscope equipped with Axiovision 4.8 software. Images were processed using contrast/brightness enhancement only. Foci were counted in at least 5 different fields totaling 50 or more cells in duplicate irradiations.

Li Z., Yu D.S., Doetsch P.W., & Werner E. (2020). Replication stress and FOXM1 drive radiation induced genomic instability and cell transformation.

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