DNA was detected by blotting the DNA with a slot blot vacuum manifold (Biorad) onto a positively charged nylon membrane. The membrane was probed with mouse anti-dsDNA (Abcam) (1:2000). Specific proteins were detected by normalizing to DNA concentration and digesting with benzonase. The proteins were separated on a polyacrylamide gel and silver stained (Sigma).
Mouse anti dsdna
Manufactured by Abcam
Sourced in Germany
Mouse anti-dsDNA is an antibody that recognizes double-stranded DNA (dsDNA). It is used for the detection and quantification of dsDNA in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Bio dot sf, by Bio-Rad (2 mentions)
Dneasy blood and tissue kit, by Qiagen (2 mentions)
Vacuum slot blot manifold, by Bio-Rad (1 mentions)
Diethanolamine substrate buffer, by Thermo Fisher Scientific (1 mentions)
P nitrophenyl phosphate substrate tablets, by Merck Group (1 mentions)
Mouse anti 5mc, by Active Motif (1 mentions)
Mouse anti 5hmc, by Active Motif (1 mentions)
Anti mouse or anti rabbit horseradish peroxidase conjugated antibody, by Abcam (1 mentions)
Mitochondrial isolation kit, by Abcam (1 mentions)
8 ohdg, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase conjugated anti rabbit antibody, by Jackson ImmunoResearch (1 mentions)
Imagequant tl software, by GE Healthcare (1 mentions)
Horseradish peroxidase conjugated anti mouse antibody, by Abcam (1 mentions)
Mouse anti 8 ohdg, by Santa Cruz Biotechnology (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
Dneasy blood tissue kit, by Qiagen (1 mentions)
8 protocols using mouse anti dsdna
1
DNA Blotting and Protein Detection
2
Enzyme-linked Immunoassay for Anti-MPO Antibodies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Nunc MaxiSorp plates were coated with 50 μl/well of 4 μg/ml anti-MPO antibody (R&D Systems) in PBS overnight at 4°C. Plates were washed three to five times with PBS/0.05% Tween and blocked with PBS/1% BSA for 1.5 h at room temperature. Plates were washed and 50 μl of undiluted plasma samples were added to each well and incubated overnight at 4°C. Plates were washed three to five times with PBS/0.05% Tween, and 50 μl of mouse anti-dsDNA (1:2,000; Abcam) diluted in 1× PBS/1% BSA was added and incubated overnight at 4°C. Plates were washed and alkaline phosphatase–conjugated anti-mouse IgG Fcγ2a (1:5,000; Jackson Immunoresearch) diluted in PBS/1% BSA was added and incubated for 3 h at 4°C, followed by development with diethanolamine substrate buffer (Thermo Fisher Scientific) and p-nitrophenyl phosphate substrate tablets (Sigma-Aldrich). Undiluted bronchoalveolar lavage fluid from mice with lung neutrophilia (Fogli et al., 2013 (link)) was used as a positive control on the same plate.
3
Detecting Top1 Cleavage Complexes by RADAR
4
Quantification of 5mC and 5hmC in Disc Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Genomic DNA was extracted and isolated from the L4–5 and L5–6 discs in each group using a DNeasy Blood and Tissue Kit (QIAGEN, Hilden, Germany) to quantify 5mC and 5hmC. Purified gDNA was spotted on a nitrocellulose membrane (0.2 µm pore size) and hybridized to the membrane by baking at 80 °C for 2 h. The membrane was then blocked with 5% skim milk and incubated with mouse anti-dsDNA (1:2000, Abcam), mouse anti-5mC (1:100, Active Motif), and mouse anti-5hmC (1:500, Active Motif) at room temperature for 1 h. Samples were then incubated with an anti-mouse or anti-rabbit horseradish peroxidase-conjugated antibody (1:1000, Abcam) for 1 h at room temperature. Antibody binding was visualized using enhanced chemiluminescence.
5
Detecting Top1 Cleavage Complexes by RADAR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The RADAR assay for TOP1cc was performed following the published protocol (Kiianitsa and Maizels, 2013 (link)). Briefly, cells were seeded in 6-well plates and treated with 2.5-10 μM CPT. After treatment, cells were lysed in 0.8 mL MB (6M GTC, 10 mM Tris-HCl, pH 6.5, 20 mM EDTA, 4% Triton X-100, 1% Sarkosyl and 1% dithiothreitol). Nucleic acids were precipitated out of the lysate by adding half volume (0.4 mL) of 100% ethanol, incubating at −20 for 5 min, and centrifuging at maximum speed for 15 min. After 2 washes in 75% ethanol, pellets were resuspended in 0.2 mL of 8 mM NaOH without drying. For immunodetection, a small portion of the lysates were diluted in Tris-buffered saline [10mM Tris (pH 7.5), 150mM NaCl] for a final volume of 0.2 mL, which was then slot blotted onto a nitrocellulose membrane with the Bio-Rad BioDot SF. Band intensities were analyzed with the online ImageJ software and normalized to the amount of loaded DNA. Antibodies used in this assay were rabbit anti-TOP1 (Bethyl, 1:2000) and mouse anti-dsDNA (Abcam, 1:2000).
6
Quantifying Oxidative DNA Damage
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We performed a DNA dot blot assay to investigate the effect of IBC on oxidative DNA damage in nuclear and mitochondrial DNA (nDNA and mtDNA). 8-OHdG, an oxidized DNA nucleoside, is the most frequently detected nucleoside in nDNA and mtDNA. In brief, nDNA was extracted and isolated from the primary cortical neurons in each group using a DNeasy Blood and Tissue Kit (QIAGEN, Hilden, Germany) to quantify 8-OHdG (n = 3 per group). mtDNA was also prepared using a mitochondrial isolation kit (Abcam plc). Purified nDNA and mtDNA samples were spotted on a nitrocellulose membrane (0.2 µm pore size) and hybridized to the membrane by baking it at 80 °C for 2 h. The membrane was then blocked with 5% skim milk and incubated with mouse anti-dsDNA (1:2000, Abcam plc) and 8-OHdG (1:200, Santa Cruz Biotechnology) at room temperature (RT) for 1 h. Samples were then incubated with an anti-mouse or anti-rabbit horseradish-peroxidase-conjugated antibody (1:1000, Jackson ImmunoResearch Laboratories) for 1 h at RT. The antibody binding was visualized based on enhanced chemiluminescence.
7
Quantifying Genomic 5hmC and 5mC
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Genomic DNA was extracted from the cerebral cortexes of the rats in each group and isolated with a DNeasy Blood (Qiagen, Hilden, Germany) and Tissue Kit (Qiagen) for quantification of 5hmC and 5mC within genomic DNA (n = 3 per group). Purified genomic DNA samples were spotted on a nitrocellulose membrane (0.2 µm pore size) using a 96-well manifold apparatus (Manifold I; Schleicher and Schuell, Dassel, Germany). The DNA was immobilized to the membrane by baking at 80 °C for 2 h. The membrane was then blocked with 5% skim milk and incubated with mouse anti-dsDNA (1:2000, Abcam), rabbit anti-5hmC (1:2000, Active Motif), or rabbit anti-5mC (1:1000, Active Motif) at room temperature (RT) for 1 h, incubated with an antimouse or antirabbit horseradish-peroxidase-conjugated antibody (Jackson) for 1 h at RT, and antibody binding was visualized by enhanced chemiluminescence (ECL). The relative amounts of 5hmC and 5mC in genomic DNA were calculated using ImageQuant TL software (GE Healthcare, v 7.0, Chicago, IL, USA) as previously described [18 (link)].
8
Quantification of 8-OHdG in Genomic DNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Genomic DNA was extracted in each group and isolated using the DNeasy Blood & Tissue Kit (Qiagen) for the quantification of the 8-OHdG amount within genomic DNA. The purified genomic DNA samples were spotted on the nitrocellulose membrane (0.2 µm pore size). The DNA was immobilized to the membrane by baking at 80 °C for 2 h. The membrane was then blocked with 5% skim milk and incubated with mouse anti-dsDNA (1:2000, Abcam) and mouse anti-8-OHdG (1:200, Santa Cruz) at RT overnight. Horseradish peroxidase-conjugated anti-mouse antibody (1:1000, Abcam) was incubated for 1 h at RT and visualized by enhanced Clarity Max Western ECL Substrate (ECL, Bio-rad). The relative amounts of 8-OHdG in the genomic DNA were calculated using Amersham™ Imager 600 (GE Healthcare Life Sciences, Little Chalfont, UK).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!