A549 cells were harvested to a density of 1.125 × 105 cells/mL in complete media and 40 µL/well seeded into 384-well plates (Greiner, Kremsmunster, Austria; 781090) using a Multidrop Combi. Following incubation under tissue culture conditions overnight, test compounds were added using an Echo 555. Following 1 h compound incubation, DNA damage was induced by the addition of 5 µL/well 9 mM H2O2 for 10 min under tissue culture conditions. Media was removed and cells were fixed in 20 µL of ice-cold methanol for 15 min at 4 °C. Blocking solution (3% bovine serum albumin [BSA] in phosphate-buffered saline with Tween 20 [PBST]) was added (1 h, RT). Twenty microliters per well of rabbit anti-PAR antibody (Trevigen, Gaithersburg, MA; 4436-BPC-100) at 1:1000 was added overnight (4 °C). Following three washes in PBST, 20 µL/well AlexaFluor 488 goat anti-rabbit (Invitrogen, 1:500) and Hoescht stain (Invitrogen, 1:5000) were added (1 h, RT). Following three washes in PBST, cells were imaged in 30 µL/well PBS using a CellInsight (Thermo Fisher) fitted with a 10× objective.
Rabbit anti par antibody
Manufactured by Bio-Techne
Sourced in United States
Rabbit anti-PAR antibody is a primary antibody that recognizes poly(ADP-ribose) (PAR), a post-translational modification found in cells. It is used as a research tool to detect and quantify PAR levels in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Cellytic pn kit, by Merck Group (3 mentions)
Par polymer, by Bio-Techne (3 mentions)
Alkaline phosphatase, by Merck Group (3 mentions)
Cellinsight, by Thermo Fisher Scientific (1 mentions)
384 well plate, by Greiner (1 mentions)
Hoescht stain, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Annexin 5, by BD (1 mentions)
Necrostatin 1, by Merck Group (1 mentions)
Nicotinamide adenine dinucleotide, by Merck Group (1 mentions)
Rabbit anti hmgb1, by Abcam (1 mentions)
Xav939, by Bio-Techne (1 mentions)
Rabbit anti caspase 3, by Cell Signaling Technology (1 mentions)
Glutaraldehyde, by Merck Group (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Z vad fmk, by Merck Group (1 mentions)
Mouse anti β actin, by Abcam (1 mentions)
Hrp conjugated anti rabbit antibody, by Thermo Fisher Scientific (1 mentions)
Mouse anti parp1 antibody, by BD (1 mentions)
5 protocols using rabbit anti par antibody
1
Detecting DNA Damage in A549 Cells
2
Plant Nuclei Isolation and PAR Quantification
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For the isolation of plant nuclei and nuclear protein extraction, the CelLytic PN kit (Sigma Aldrich) was used [109 (link)]. The protein (5 μg) was analysed for PAR accumulation levels by ELISA using (Trevigen) purified monoclonal antibody to PAR as the capture reagent, a rabbit anti-PAR antibody (Trevigen) as the detecting agent, and a goat anti-rabbit antibody conjugated with alkaline phosphatase (Sigma Aldrich) as the reporter [110 (link)]. PAR polymer (Trevigen) was used as a positive control.
3
Cell Death Pathway Analysis
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The following reagents were obtained commercially: rabbit anti-caspase-3 from Cell Signaling Technology (Danvers, MA, USA); rabbit anti-HMGB1 from Abcam (Cambridge, UK); mouse anti-β-actin, H2O2, t-BHP, propidium iodide (PI), STS, Necrostatin-1 (Nec-1), 3-Aminobenzamide (3AB), 3,4-dihydro-5-[4-(1-piperidinyl)butoxyl]-1(2H)-isoquinolinone (DPQ), poly-l -lysine, nicotinamide adenine dinucleotide (NAD), SI and 50% glutaraldehyde from Sigma-Aldrich (Saint Louis, MO, USA); horseradish peroxidase (HRP)-conjugated anti-mouse antibody, HRP-conjugated anti-rabbit antibody, fluorescein (FITC)-conjugated anti-mouse antibody and 4′,6-diamidino-2-phenylindole (DAPI) from Thermo Fisher Scientific (Rockford, IL, USA); mouse anti-RIPK1 antibody, mouse anti-PARP-1 antibody, and Annexin V from BD Biosciences (San Jose, CA, USA); rabbit anti-RIPK3 antibody and mouse anti-AIF antibody from Santa Cruz Biotechnology (Dallas, TX, USA); rabbit anti-PAR antibody from Trevigen (Gaithersburg, MD, USA); mouse anti-PAR antibody from Enzo Life Sciences (Farmingdale, NY, USA); z-VAD-fmk from Millipore (Darmstadt, Germany); olaparib from Selleck Chemicals (Houston, TX, USA); UPF-1069 and XAV-939 from Tocris Bioscience (Minneapolis, MN, USA); and MNNG from Tokyo Chemical Industry (Tokyo, Japan).
4
Plant Nuclei Isolation and PAR Quantification
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For the isolation of plant nuclei and nuclear protein extraction the CelLytic PN kit (Sigma-Aldrich, St. Louis, MO, USA) was used [61 (link)]. The protein (5 µg) was analysed for PAR accumulation levels by ELISA using a purified monoclonal antibody (Trevigen, Gaithersburg, MD, USA) to PAR as the capture reagent, a rabbit anti-PAR antibody (Trevigen, Gaithersburg, MD, USA) as the detecting agent, and a goat anti-rabbit antibody conjugated with alkaline phosphatase (Sigma-Aldrich, St. Louis, MO, USA) as the reporter [62 (link)]. PAR polymer (Trevigen, Gaithersburg, MD, USA) was used as a positive control.
5
Plant Nuclear Protein Extraction and PAR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
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For the isolation of plant nuclei and nuclear protein extraction, the CelLytic PN kit (Sigma Aldrich) was used [41 (link)]. The protein (5 µg) was analysed for PAR accumulation levels by ELISA using purified monoclonal antibody (Trevigen) to PAR as the capture reagent, a rabbit anti-PAR antibody (Trevigen; Gaithersburg, MD, USA) as the detecting agent, and a goat anti-rabbit antibody conjugated with alkaline phosphatase (Sigma Aldrich) as the reporter [42 (link)]. PAR polymer (Trevigen) was used as a positive control.
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