Rpa32

Manufactured by Fortis Life Sciences

Sourced in Morocco, United States

The RPA32 is a laboratory equipment designed for performing automated liquid handling tasks. It features a robotic arm that can precisely pipette and transfer liquids between vessels, enabling efficient and consistent liquid handling operations in various laboratory workflows.

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Lab products found in correlation

Imagequant las 500, by GE Healthcare (3 mentions) Rpa32 s4 s8, by Fortis Life Sciences (3 mentions) Rad51, by Santa Cruz Biotechnology (3 mentions) Proliferating cell nuclear antigen (pcna), by Santa Cruz Biotechnology (2 mentions) Bradford assay, by Bio-Rad (2 mentions) Lamin a c, by Cell Signaling Technology (2 mentions) Protease and phosphatase inhibitor cocktail, by Roche (2 mentions) Rpa32, by Thermo Fisher Scientific (1 mentions) Topbp1, by Santa Cruz Biotechnology (1 mentions) Rad52, by Santa Cruz Biotechnology (1 mentions) Parp 1, by Santa Cruz Biotechnology (1 mentions) Flag m2, by Merck Group (1 mentions) Mre11, by Novus Biologicals (1 mentions) Ha tag, by Santa Cruz Biotechnology (1 mentions) Hnrnpd, by Merck Group (1 mentions) Hnrnpd, by Cell Signaling Technology (1 mentions) Chk1 s345, by Cell Signaling Technology (1 mentions) Novex ecl chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions) Xrcc4, by Santa Cruz Biotechnology (1 mentions) Cleaved capsase 3, by Cell Signaling Technology (1 mentions)

Close spelling variants of this product

Anti-RPA32 (9 citations) Anti-phospho-RPA32 (S4/S8) (5 citations) Phospho-RPA32/S4S8 (5 citations) RPA32 pS4/8 (4 citations) Anti-phospho-RPA32 (4 citations) RPA32 S4/S8 (3 citations) RPA32 S4/8 (2 citations) Anti-phospho RPA32 (S33) (2 citations) Phospho RPA32(S33) (2 citations) Phosphorylated RPA32 (S4/S8) (2 citations)

10 protocols using rpa32

Immunoblotting Analysis of DNA Damage Response

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Immunoblotting analysis was performed as described previously (27–29 (link),60 (link),64 (link)). Primary antibodies were purchased from respective vendors: APE1 (Santa Cruz Biotechnology Cat#sc-17774), ATR (Santa Cruz Biotechnology Cat#515173), ATM (GeneTex Cat#GTX70103), ATM phosphorylation at Ser1981 (Abcam Cat#ab81292), ATR phosphorylation at Thr1989 (Cell Signaling Technology Cat#D5K8W), ATRIP (Santa Cruz Biotechnology Cat#365383), Chk1 (Santa Cruz Biotechnology Cat#sc-8408), Chk1 phosphorylation at Ser345 (Cell Signaling Technology Cat#133D3), Chk1 phosphorylation at Ser317 (Cell Signaling Technology Cat#D12H3), Chk2 (Santa Cruz Biotechnology Cat#sc-9064), Chk2 phosphorylation at Thr68 (Santa Cruz Biotechnology Cat#sc-16297), eGFP (Life Technologies Corporation Cat#TA150041), ETAA1 (Abcam Cat#ab122245), H2AX (Cell Signaling Technology Cat#2D17A3), H2AX phosphorylation at Ser139 (Cell Signaling Technology Cat#2577s), NPM1 (Santa Cruz Biotechnology Cat#sc-47725), p53 phosphorylation at Ser15 (Cell Signaling Technology Cat#9284), p53 (Santa Cruz Biotechnology Cat#sc-126), PCNA (Santa Cruz Biotechnology Cat#sc-56), RPA32 (Thermo Fisher Scientific Cat#MA1-26418), RPA32 phosphorylation at Ser33 (Bethyl Laboratories Cat#A300-246A), TopBP1 (Santa Cruz Biotechnology Cat#271043), Tubulin (Santa Cruz Biotechnology Cat#sc-8035), YFP (BioVision Cat#3991–100).

Li J., Zhao H., McMahon A, & Yan S. (2022). APE1 assembles biomolecular condensates to promote the ATR–Chk1 DNA damage response in nucleolus. Nucleic Acids Research, 50(18), 10503-10525.

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Western Blot Analysis of DNA Repair Proteins

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The following antibodies were used: RPA32 (A300-244A, Bethyl Laboratories), RPA32 S4/S8 (A300-245A, Bethyl Laboratories), Lamin A/C (#4777, Cell Signalling), RAD52 (sc-365341, Santa Cruz Biotechnology), RAD51 (sc-8349, Santa Cruz Biotechnology), ERCC1 (NB500-704, Novus Biological), RAD52 (sc-365341, Santa Cruz Biotechnology), PARP1 (sc-8007, Santa Cruz Biotechnology). For total protein extraction, cells were lysed at 4°C in 50 mM HEPES pH7.5, 1% Triton X-100, 150 mM NaCl, 5 mM EGTA, supplemented with protease and phosphatase inhibitor cocktail (Roche Applied Science). Lysates were clarified by centrifugation at 10.000 × g for 20 min. Lysates containing equal amounts of proteins, estimated through the Bradford assay (Bio- Rad), were subjected to SDS-page. The chemiluminescent images were obtained using the ImageQuant LAS 500 (GE Healthcare). Band densitometry values inserted in all western blot figures indicate the levels of all phosho-proteins normalized to correspondent total protein and the last are normalized with the internal reference protein.

Barone D., Iannuzzi C.A., Forte I.M., Ragosta M.C., Cuomo M., Dell’Aquila M., Altieri A., Caporaso A., Camerlingo R., Rigano M.M., Monti D.M., Barone A., Imbimbo P., Frusciante L., Monda M., D’Angelo M., De Laurentiis M., Giordano A, & Alfano L. (2023). The hydrophilic extract from a new tomato genotype (named DHO) kills cancer cell lines through the modulation of the DNA damage response induced by Campthotecin treatment. Frontiers in Oncology, 13, 1117262.

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Antibody Detection and Protein Extraction

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The following antibodies were used: HNRNPD (1:1000, 07-260, Millipore, RRID:AB_2117338), HNRNPD (1:1000, D6O4F, Cell Signalling, Danvers, MA, RRID:AB_2616009), RPA32 (1:5000, A300–244A, Bethyl Laboratories, RRID:AB_185548), RPA32 S4/S8 (1:2000, A300–245A, Bethyl Laboratories), H3 (1:1000, #9715, Cell Signalling, RRID:AB_331563), CHK1 S345 (1:1000, #2348, Cell Signalling), CHK1 (1:1000, #2360, Cell Signalling), GAPDH (1:1000, sc-25778, Santa Cruz, Dallas), MRE11 (1:1000, NB100–142, Novus Biological), EXOI (1:1000, A302–640A, Bethyl Laboratories), CtIP (1:1000, #61142, Active Motif) SAF-A (1:1000, ab10297, Abcam), RAD17 S645 (1:2000, ab3620, Abcam), FLAG-M2 (1:1000, F1804, SIGMA Aldrich), HA-tag (1:500, sc-805, Santa Cruz Biotechnology), Lamin A/C (1:1000, #4777, Cell Signalling), GFP (1:5000, ab6556, Abcam), His-tag (1:1000, 05-531, Millipore). For total protein extraction, cells were lysed at 4°C in 50 mM HEPES pH7.5, 1% Triton X-100, 150 mM NaCl, 5 mM EGTA, supplemented with protease and phosphatase inhibitor cocktail (Roche Applied Science). Lysates were clarified by centrifugation at 10 000 × g for 20 min. Lysates containing equal amounts of proteins, estimated through the Bradford assay (Bio-Rad), were subjected to SDS-page. The chemiluminescent images were obtained using the ImageQuant LAS 500 (GE Healthcare).

Alfano L., Caporaso A., Altieri A., Dell’Aquila M., Landi C., Bini L., Pentimalli F, & Giordano A. (2019). Depletion of the RNA binding protein HNRNPD impairs homologous recombination by inhibiting DNA-end resection and inducing R-loop accumulation. Nucleic Acids Research, 47(8), 4068-4085.

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Radiation-Induced Apoptosis and DNA Repair Signaling

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Cells were irradiated at 70–90% confluence, and lysates were prepared at specific time points post-radiation injury. Cells were washed three times with PBS and then extracted with RIPA buffer (50 mM Tris (pH 8), 150 mM NaCl, 0.1% SDS, 1% NP40, 0.5% sodium deoxycholate and ×1 Halt Protease and phosphatase inhibitor (cat# 78443, Thermofisher, Rockville MD). Primary antibodies were obtained for full-length caspase 3 (cat. #9662S, Cell Signaling,, Danvers, MA) (1:1000), cleaved capsase 3 (cat. #9661S, Cell Signaling) (1:1000), and p21 (cat. #9sc-397, Santa Cruz Biotechnology, Inc., Dallas TX) (1:500), p-RPA32 (cat. #A300–245A, Bethyl, Montgomery, TX) (1:500), RPA32 (cat#A300–244A, Bethyl) (1:500), Rad51 (cat. #98875S, Cell Signaling) (1:500), Ku70 (cat. #9sc-9033, Santa Cruz Biotechnology) (1:1000), and XRCC4 (cat. #sc-8285, Santa Cruz Biotechnology) (1:300). Proteins were detected with species-matched horseradish peroxidase–linked secondary antibodies (1:500–1:2000, R&D Systems) and Novex ECL Chemiluminescent Substrate (Cat # WP20005, Thermofisher). WCIF ImageJ software was used for densitometry analysis (NIH, Bethesda MD; https://imagej.nih.gov/ij/download.html).

Bylicky M.A., Mueller G.P, & Day R.M. (2018). Radiation resistance of normal human astrocytes: the role of non-homologous end joining DNA repair activity. Journal of Radiation Research, 60(1), 37-50.

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Investigating Cell Cycle Regulation

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The following chemical inhibitors were used at the indicated concentrations unless stated otherwise: WEE1 inhibitor MK-1775 (500 nM; Selleck Chemicals), CHK1 inhibitor SB 218078 (2 μM; Tocris), ATR inhibitor VE-821 (20 μM; Selleck Chemicals), CDK1 inhibitor RO-3306 (10 μM; Calbiochem), CDK inhibitor Roscovitine (20 μM; Selleck Chemicals), thymidine (2 mmol/L; Sigma-Aldrich), and nocodazole (100 ng/mL; Sigma-Aldrich), IdU (10 μg/ml, Sigma-Aldrich), CIdU (10 μg/ml, Sigma-Aldrich). The following antibodies were used: CHK1 (Santa Cruz Biotechnology), CHK1-pS345 (Cell Signaling), CHK2 (Santa Cruz Biotechnology), CHK2-pT68 (Cell Signaling), phospho-Histone-H2AX-Ser139 (γH2AX; Millpore), CDK1 (Santa Cruz Biotechnology), CDK1-pTyr15 (Cell Signaling), RPA70 (Abcam), phospho-(Ser) CDKs Substrate (pCDK Substrate; Cell Signaling), CDK2 (Santa Cruz Biotechnology), CDK2-pTyr15 (Abcam), RPA32-pT21 (Abcam), RPA32-pS33 (Abcam), RPA32-pS4/8 (Bethyl Laboratories), RPA32 (Bethyl Laboratories), WEE1 (Santa Cruz Biotechnology), α-Tubulin (Sigma-Aldrich), CIdU (rat anti-BrdU; Accurate Chemical), IdU (mouse anti-BrdU; Becton Dickinson).

Zheng H., Shao F., Martin S., Xu X, & Deng C.X. (2017). WEE1 inhibition targets cell cycle checkpoints for triple negative breast cancers to overcome cisplatin resistance. Scientific Reports, 7, 43517.

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Western Blot Analysis of DNA Damage Response

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For total protein extraction, cells were lysed on ice for 30 min in a buffer consisting of 50 mM Tris-HCl pH 7.5, 1 mM EDTA pH 8.0, 150 mM NaCl, 1% NP-40, supplemented with protease and phosphatase inhibitor cocktails (Roche, Basilea, Switzerland). The protein samples were resolved by SDS-PAGE and blotted onto nitrocellulose membranes, which were then incubated with antibodies against: ɣ-H2AX (Cat. #05-636) from Merk Millipore, Burlington, MA, USA; phospho-RPA32 Ser 4/Ser 8 (Cat. #A300–245A) and RPA32 (Cat. #A300–244A) from Bethyl Laboratories, Montgomery, TX, USA; phospho-CHK1 Ser 345 (Cat. #2348), CHK1 (Cat. #2360), phospho-CDK1 Tyr15 (Cat. #4539), CDK1 (Cat. #9116S), and caspase-3 (Cat. #9662S) from Cell Signaling Technologies, Danvers, MA, USA; BCL-XS/L (Cat. #sc-1041) and GAPDH (Cat. #sc-25778) from Santa Cruz Biotechnology, Santa Cruz, CA, USA. After incubation with horseradish peroxidase-conjugated secondary antibodies, signals were detected through ECL (Amersham Biosciences, GE Healthcare, Little Chalfont, UK). The chemiluminescent images were analyzed by ImageQuant LAS 500 (GE Healthcare, Little Chalfont, UK).

Iannuzzi C.A., Indovina P., Forte I.M., Di Somma S., Malfitano A.M., Bruno M., Portella G., Pentimalli F, & Giordano A. (2020). Pharmacological Inhibition of WEE1 Potentiates the Antitumoral Effect of the dl922-947 Oncolytic Virus in Malignant Mesothelioma Cell Lines. International Journal of Molecular Sciences, 21(19), 7333.

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Western Blot Analysis of Chromatin Proteins

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MEFs were lysed in RIPA buffer with protease and phosphatase inhibitors and lysates were processed using standard methods. We used the following primary antibodies: VHL (sc-5575, 1:250; Santa Cruz), PBRM1 (A301-590A, 1:1000; Bethyl Laboratories), alpha-Tubulin (T5168, 1:2000; Sigma), RAD51 (sc-8349, 1:100; Santa Cruz), RPA32 (A300-244A, 1:500; Bethyl Laboratories), pATM (ab36810, 1:1000; Abcam), ATM (ab85213, 1:1000; Abcam), pDNA-PKc (ab18192, 1:1000; Abcam), DNA-PKc (ab70250, 1:1000; Abcam), pCHK1 (#2348 S, 1:500; Cell Signalling), CHK1 (#2360, 1:1000; Cell Signalling), H3K9me3 (ab8898, 1:1000; Abcam), H3K27me3 (ab6002, 1:1000; Abcam), H3K9me2 (ab8898, 1:1000; Abcam), H4K20me3 (ab9053, 1:1000; Abcam), HP1a (ab77256, 1:1000, Abcam), KAP1 (ab10484, 1:1000; Abcam) and H3 (4499, 1:2000; Cell Signalling). Secondary antibodies were conjugated to IRDye 680 or 800 (Li-Cor). Fluorescent signals were imaged using the Odyssey Infrared Imaging System (Li-Cor). Western blot band quantifications were performed with Fiji^{58 (link)}. Uncropped scans of presented blots are shown in Supplementary Fig. 7.

Espana-Agusti J., Warren A., Chew S.K., Adams D.J, & Matakidou A. (2017). Loss of PBRM1 rescues VHL dependent replication stress to promote renal carcinogenesis. Nature Communications, 8, 2026.

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DNA Damage Response Protein Analysis

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Cells were lysed with mammalian protein extraction reagent (ThermoFisher Scientific) and equivalent protein lysates were subjected to SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane. Immunoblots were developed using Pierce enhanced chemiluminescence reagents and Bio-Max film (ThermoFisher Scientific). Antibodies used were γH2AX, (Millipore, 05–636); pATR (S428), (Cell Signaling, 2853; Danvers, MA, https://www.cellsignal.com); RPA32, (Bethyl Laboratories, A300–244A; Montgomery, Texas, https://www.bethyl.com); RPA32-S33, (Bethyl Laboratories, A300–246A; RPA32-S4/S8, Bethyl Laboratories, A300–245A); pCHK1 (S345), (Cell Signaling, 2341); RAD51, (Santa Cruz Biotechnology, sc-8349); p53-S15, (Cell Signaling, 9286; Actin, Sigma Aldrich, A5316; St. Louis, MO, https://www.sigmaaldrich.com; CHK1, Cell Signaling, 2360); ATR, (Cell Signaling, 2790); CHK2, (Cell Signaling, 2662); ATM, (Abcam, ab78), Cyclin A, (Cell Signaling, 4656); CDK2, (Cell Signaling, 2546); and γH2AX, (Cell Signaling, 9718).

Vallabhaneni H., Lynch P.J., Chen G., Park K., Liu Y., Goehe R., Mallon B.S., Boehm M, & Hursh D.A. (2018). High Basal Levels of γH2AX in Human Induced Pluripotent Stem Cells Are Linked to Replication-Associated DNA Damage and Repair. Stem cells (Dayton, Ohio), 36(10), 1501-1513.

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Characterization of DNA Damage Response

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hPFs were from Dr. Prudence Talbot (UC Riverside) and transformed nontumorigenic human lung epithelial cells (BEAS-2B) were from ATCC (Manassas, Virginia). Normal human skin fibroblasts (HCA2) were from J. Smith (University of Texas, USA) and were immortalized by infection with an hTERT expressing retrovirus [Rubio et al. 2002 (link)]. XPA deficient patient skin fibroblasts XP12BE were from Coriell (Camden, NJ).
Antibodies used were 53BP1 (A300–272A, Bethyl), phospho-RPA32 (S4/S8: A300–245A, Bethyl), RPA32 (A300–244A, Bethyl), phospho-H2AX S139-clone JBW301 (EMD Millipore), CHK1 (2345, Cell Signaling), pCHK1-Ser317 (2344S, Cell Signaling), pATM (S1981, Ab81292, Abcam), ATM (Cell Signaling), PCNA (Santa Cruz), pBRCA1 (S1423, A300–008A, Bethyl), pATR (Thr1989, GTX128145, GeneTex), Tubulin (ab4074, Abcam) and GAPDH (MAB374, EMD Millipore) and rabbit anti-XPA (sc853, Santa Cruz).

Sarker A.H., Trego K.S., Zhang W., Jacob P I.I.I., Snijders A., Mao J.H., Schick S.F., Cooper P.K, & Hang B. (2020). Thirdhand Smoke Exposure Causes Replication Stress and Impaired Transcription in Human Lung Cells. Environmental and molecular mutagenesis, 61(6), 635-646.

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Western Blotting Antibody Protocol

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Antibodies for western blotting purchased from Cell signaling (Danvers, MA): phospho-CHK1 (S345) (2348, 1:1000), CHK1 (2360, 1:1000), γH₂A_x (9718, 1:1000), AXL (8661, 1:1000), Cyclin B1 (4138), phospho-cdc2 (Y15) (4539), phospho-Histone H3 (S10) (53348), phospho-AKT (S473) (4060), AKT (9272), phospho-S6 (S240/244) (2215), S6 (2217), p21 (2946); Bethyl laboratories : RPA32 (A300–244, 1:1000), phospho-RPA32 (S4/S8) (A300–245, 1:1000), phospho-RPA32 (S33) (A300–246, 1:1000) and phospho-KAP1 (S824) (A300–246, 1:1000); Santa Cruz Biotechnology (Dallas, TX) : β-actin (sc-47778, 1:2000), GAPDH (sc-20357, 1:2000). BGB324 was generously provided by BergenBio and AZD6738 by AstraZeneca. VX-970 was purchased from Selleck Chemicals (Houston, TX). Vinculin (V9131), Propidium iodide and Hydroxyurea were purchased from and Sigma-Aldrich (St. Louis, MO), respectively.

Ramkumar K., Stewart C.A., Cargill K.R., Corte C.M., Wang Q., Shen L., Diao L., Cardnell R.J., Peng D.H., Rodriguez B.L., Fan Y.H., Heymach J.V., Wang J., Gay C.M., Gibbons D.L, & Byers L.A. (2020). AXL inhibition induces DNA damage and replication stress in non-small cell lung cancer cells and promotes sensitivity to ATR inhibitors. Molecular cancer research : MCR, 19(3), 485-497.

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