Rabbit polyclonal anti parp

Manufactured by Cell Signaling Technology

Sourced in United States

Rabbit polyclonal anti-PARP is a laboratory reagent that can be used to detect the presence of the PARP protein in biological samples. It is a polyclonal antibody produced in rabbits, which means it recognizes multiple epitopes on the PARP protein.

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7 protocols using rabbit polyclonal anti parp

Western Blot Analysis of Cellular Proteins

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Cells were washed twice in ice-cold PBS and immediately lysed in Laemmli buffer as described previously [38 (link)]. Equal amounts of cell lysates were resolved by 10% SDS/PAGE and electro-transferred onto poly(vinylidene difluoride) membrane. Immunoblot analyses were performed as described previously [19 (link)] using the primary antibodies: mouse monoclonal anti-p53 (DO-1; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse monoclonal anti-p73 (Ab-4; NeoMarkers, Fremont, CA, USA), mouse monoclonal anti-γH2AX (2F3; BioLegend, San Diego, CA, USA), rabbit polyclonal anti-p21^WAF1 (Santa Cruz Biotechnology), rabbit polyclonal anti-phospho-p53 at Ser-15 (Cell Signaling Technology, Beverly, MA, USA), rabbit polyclonal anti-RUNX2 (Cell Signaling Technology), rabbit polyclonal anti-E2F-1 (Cell Signaling Technology), rabbit polyclonal anti-PARP (Cell Signaling Technology), rabbit polyclonal anti-caspase-9 (Cell Signaling Technology) and rabbit polyclonal anti-Actin (20–33; Sigma, St Louis, MO, USA) antibodies. Immunoreactive bands were visualized by an enhanced chemiluminescence system (ECL; GE Healthcare, Little Chalfont, UK) in accordance with the manufacturer's instructions.

Ozaki T., Sugimoto H., Nakamura M., Hiraoka K., Yoda H., Sang M., Fujiwara K, & Nagase H. (2014). Runt-related transcription factor 2 attenuates the transcriptional activity as well as DNA damage-mediated induction of pro-apoptotic TAp73 to regulate chemosensitivity. The Febs Journal, 282(1), 114-128.

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Immunoblotting and Immunofluorescence Assays

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Immunoblotting and immunofluorescence were performed as described [22 (link), 23 (link)]. FGF19 protein levels in the medium were determined using human FGF-19 Quantikine ELISA Kit (R&D Systems). The following antibodies were used: mouse monoclonal anti-BrdU (BD Pharmingen); mouse monoclonal anti-Ki-67 (BD Pharmingen); mouse monoclonal anti-nucleolin (C23, Santa Cruz Biotechnology); rabbit polyclonal anti-PARP (#9542, Cell Signaling); mouse monoclonal anti-phospho-ERK (E4, Santa Cruz Biotechnology); rabbit polyclonal anti-phospho-FRS2 (Tyr196) (#3864, Cell Signaling); mouse monoclonal anti-FRS2 (MAB4069, R&D Systems); and mouse monoclonal anti-RB (G3-245, BD Pharmingen).

Elzi D.J., Song M., Blackman B., Weintraub S.T., López-Terrada D., Chen Y., Tomlinson G.E, & Shiio Y. (2016). FGF19 functions as autocrine growth factor for hepatoblastoma. Genes & Cancer, 7(3-4), 125-135.

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Protein Isolation and Western Blot

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Cells were treated with vehicle, quarfloxin, or CX-5461 as indicated. At harvesting, floating cells and adherent cells (trypsination) were collected for analysis. Protein isolation and blotting were performed essentially as previously described using NuPAGE 4–12% bis-tris precast polyacrylamide gels and Immobilon-PVDF membranes (Millipore) [42 (link)]. For western blots containing Caspase-3 and p21, 4–20% Tris-Glycine gels (Lonza) were used for better separation of these low MW proteins. Primary antibodies used in this study were mouse monoclonal anti-MycN (sc-53993, Santa Cruz Biotechnology, CA, USA), mouse monoclonal anti-p53 (sc-126, Santa Cruz Biotechnology, CA, USA), rabbit monoclonal anti-p21 (#2947, Cell Signaling Technology, MA, USA), rabbit polyclonal anti-PARP (#9542, Cell Signaling Technology, MA, USA), rabbit polyclonal anti-Caspase-3 (#9662, Cell Signaling Technology, MA, USA), mouse monoclonal anti-γ-H2A.X (05–636, Merck Millipore, MA, USA), rabbit polyclonal anti-actin (A2066, Sigma-Aldrich, MO, USA) and mouse monoclonal anti-actin (AB3280, Abcam, Cambridge, UK). Membranes were detected using the Odyssey Infrared Imaging system (LI-COR).

Hald Ø.H., Olsen L., Gallo-Oller G., Elfman L.H., Løkke C., Kogner P., Sveinbjörnsson B., Flægstad T., Johnsen J.I, & Einvik C. (2018). Inhibitors of ribosome biogenesis repress the growth of MYCN-amplified neuroblastoma. Oncogene, 38(15), 2800-2813.

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Antibody Sources for Signaling Pathways

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Antibodies were purchased from the following resources: mouse monoclonal anti-Cathepsin K (clone 182-12G5) (Millipore); rat anti-Notch2 (clone C651.6DbHN) (Developmental Studies Hybridoma Bank); rabbit polyclonal anti-ERK1/2, mouse monoclonal anti-phospho-ERK1/2 (Thr202/Tyr204), mouse monoclonal anti-Akt (pan) (clone 40D4), rabbit monoclonal anti-phosphor-Akt (Ser473) (clone 193H12), rabbit polyclonal anti-JNK, mouse monoclonal anti-phospho-JNK (Thr183/Tyr185) (clone G9), rabbit polyclonal anti-IKB-α, mouse monoclonal anti-phospho-IKB-α (Ser32/36) (clone 5A5), rabbit monoclonal anti-cleaved caspase-3 (catalog number 9664), rabbit polyclonal anti-PARP (catalog number 9542), rabbit monoclonal anti-Numb (C29G11) (catalog number 2756) (Cell Signaling Technology); mouse monoclonal anti-α-tubulin (clone DM1A) (Sigma-Aldrich); mouse monoclonal anti-beta actin (catalog number A00702) (GenScript).
Cell culture alpha-MEM and 10 × Penicillin-Streptomycin-L-Glutamine were purchased from Life Technologies and Sigma-Aldrich, respectively. Fetal bovine serum was purchased from Hyclone.

Zhou J., Fujiwara T., Ye S., Li X, & Zhao H. (2015). Ubiquitin E3 Ligase LNX2 is Critical for Osteoclastogenesis in vitro by Regulating M-CSF/RANKL Signaling and Notch2. Calcified tissue international, 96(5), 465-475.

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Antibody-based Western Blotting Analysis

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Western blotting analysis was performed as the previous study.^{19 (link)} Antibodies used were as following: rabbit polyclonal anti-PARP, rabbit polyclonal anti-caspase-3, rabbit polyclonal anti-cleaved caspase-3, rabbit polyclonal anti-caspase-9, rabbit polyclonal anti-cleaved caspase-9, rabbit polyclonal anti-cyclin B1, mouse polyclonal anti-cdc2 and rabbit polyclonal anti-p-cdc2 (Tyr15) were purchased from Cell Signaling Technology (Beverly, MA, USA). Mouse polyclonal anti-MPM2 and rabbit polyclonal anti-BICD2 were commercially available from Millipore Corporation (Bedford, MA, USA), and rabbit polyclonal anti-FOXM1 antibody was purchased from ABclonal Technology (Wuhan, China). Mouse polyclonal anti-β-actin was purchased from Sunshine Biotechnology Ltd. Goat polyclonal anti-rabbit IgG conjugated to HRP and goat polyclonal anti-mouse IgG conjugated to HRP (Cell Signaling Technology) were used as secondary antibodies, and enhanced chemiluminescence reagents (Millipore) was used for detection and exposed by Gel 2000 image analyzer (Bio-Rad, Richmond, CA, USA).

Li H., Sun L., Li H., Lv X., Semukunzi H., Li R., Yu J., Yuan S, & Lin S. (2017). DT-13 synergistically enhanced vinorelbine-mediated mitotic arrest through inhibition of FOXM1-BICD2 axis in non-small-cell lung cancer cells. Cell Death & Disease, 8(5), e2810-.

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Cloning and Characterization of HSP25 Mutants

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Wildtype (WT) mouse HSP25 (GenBank accession number, NM_013560.2) was polymerase chain reaction (PCR)-amplified, and the dimerization mutant HSP25 (C141A) was constructed by overlap extension PCR. The PCR fragments were digested with BamHI-EcoRI and ligated into p3x-Flag-Myc-CMV-26, yielding p3x-flag-HSP25 (WT) and p3x-flag-HSP25 (C141A). WT and dimerization mutant (C141A) HSP25 were cloned into p3x-Flag-myc-cmv-26 expression vector [21 (link)]. Goat polyclonal anti-HSP27 (sc-1049), goat polyclonal anti-HSP70 (sc-1060), mouse monoclonal anti-HSP90α/β (sc-13119), mouse monoclonal anti-HSF1 (sc-17757), rabbit-polyclonal anti-PKCδ (sc-213) and mouse monoclonal anti-β-actin (sc-47778) antibodies were purchased from Santa Cruz Biotechnology. Rabbit polyclonal anti-cleaved caspase-3 (#9661), rabbit polyclonal anti-cleaved PARP (#9541), and rabbit polyclonal anti-cleaved cytochrome c (#9661) antibodies were purchased from Cell Signaling (Beverly, MA, USA). Mouse monoclonal anti-cytochrome c (#556432, BD Biosciences) and rabbit polyclonal anti-PARP (#9542, Cell signaling) antibodies were used for immuneprecipitation. Mouse-monoclonal anti-HSP27 (ADI-SPA-800, Enzo) antibody was also used for immunohistochemistry.

Kim J.H., Jung Y.J., Choi B., Lee N.L., Lee H.J., Kwak S.Y., Kwon Y., Na Y, & Lee Y.S. (2016). Overcoming HSP27-mediated resistance by altered dimerization of HSP27 using small molecules. Oncotarget, 7(33), 53178-53190.

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Western Blot Analysis of Protein Levels

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Protein concentration was determined using the Bio-Rad D_C protein assay (Bio-Rad Laboratories). 20 µg of total protein were electrophoresed in 8% SDS-PAGE gel and transferred to Immobilon-P Membrane, PVDF (Millipore). Membranes were incubated overnight at 4°C with primary antibodies. Rabbit polyclonal anti-PSMC3IP, Sigma-Aldrich (HPA044439) at dilution (1:1000), rabbit polyclonal anti-EPSTI1, Sigma Aldrich (SAB2100696) at dilution (1:2000), both from Sigma Aldrich, rabbit polyclonal anti-PARP, Cell Signaling Technology (#9542) at dilution (1:1000) and mouse monoclonal anti-β-actin, Abcam (ab20272), at dilution (1:20000) followed by incubation with the appropriated HRP-conjugated secondary antibody plus enhanced chemiluminescence substrate (GE Healthcare). Protein band amounts were roughly quantified by densitometry using ImageJ (http://rsb.info.nih.gov/ij/), following standard procedures. Loading control protein β-actin was used as a reference to compare relative protein amounts.

Capdevila-Busquets E., Badiola N., Arroyo R., Alcalde V., Soler-López M, & Aloy P. (2015). Breast Cancer Genes PSMC3IP and EPSTI1 Play a Role in Apoptosis Regulation. PLoS ONE, 10(1), e0115352.

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