The reactions analyzing mono-ADP ribosylation were performed essentially as described by Liszt et al., (2005) (link). Reactions contained 5 µg hSIRT6 WT or mutant proteins, 5 µg of the truncated PARP1 missing catalytic domain (C-terminal truncation; containing only amino acid [aa] 1–655), or truncated PARP1 harboring K521A, 50 mM Tris$Cl, 150 mM NaCl, 10 mM DTT, 1 µM unlabeled NAD+, and 8 µCi 32P NAD+ (PerkinElmer, NEG023X500UC) in a 50-µl volume. Auto-ribosylation reactions by hSIRT6 and its mutants were carried out at 37°C for 30 min, and ribosylation reactions on catalytically inactive PARP1 by hSIRT6 were kept at 37°C for 2 hr. After reactions were completed, the samples were 2,2,2-trichloroacetic acid (TCA) precipitated to remove unincorporated 32P NAD+. The purified samples were loaded on SDS-PAGE gels, Commassie stained, dried, and subjected to autoradiography. Reactions analyzing activation of PARP1 by human SIRT6 contained 2 µl PARP1 (Sigma, P0996), 5 µg SIRT6WT ormutant proteins, 20 mM Tris$Cl (pH 8.0), 100 mM NaCl, 10 mM MgCl2, 10 µM ZnCl2, 10% glycerol, 300 µM NAD+, 1 mM DTT, and 0.1 µg/ml sonicated salmon sperm DNA in a 50-µl volume. These reactions were performed at 30°C for 30 min. The samples were then analyzed by western blotting using anti-PAR antibody (BD Pharmigen, 550781).
Anti par antibody
Manufactured by BD
The Anti-PAR antibody is a laboratory reagent used for the detection and quantification of poly(ADP-ribose) (PAR) in biological samples. PAR is a post-translational modification that plays a role in various cellular processes, including DNA repair and cell signaling. The Anti-PAR antibody can be used in techniques such as immunohistochemistry, Western blotting, and enzyme-linked immunosorbent assay (ELISA) to analyze the presence and levels of PAR in cells or tissues.
Automatically generated - may contain errors
Lab products found in correlation
Neg023x500uc, by PerkinElmer (2 mentions)
P0996, by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Anti tbparp antibody, by GenScript (2 mentions)
Bx41 microscope, by Olympus (1 mentions)
Alexa fluor 594 goat anti rabbit igg antibody, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti rabbit igg antibody, by Thermo Fisher Scientific (1 mentions)
5 protocols using anti par antibody
1
Quantifying SIRT6-Mediated ADP-Ribosylation
2
Quantifying SIRT6-Mediated ADP-Ribosylation
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The reactions analyzing mono-ADP ribosylation were performed essentially as described by Liszt et al., (2005) (link). Reactions contained 5 µg hSIRT6 WT or mutant proteins, 5 µg of the truncated PARP1 missing catalytic domain (C-terminal truncation; containing only amino acid [aa] 1–655), or truncated PARP1 harboring K521A, 50 mM Tris$Cl, 150 mM NaCl, 10 mM DTT, 1 µM unlabeled NAD+, and 8 µCi 32P NAD+ (PerkinElmer, NEG023X500UC) in a 50-µl volume. Auto-ribosylation reactions by hSIRT6 and its mutants were carried out at 37°C for 30 min, and ribosylation reactions on catalytically inactive PARP1 by hSIRT6 were kept at 37°C for 2 hr. After reactions were completed, the samples were 2,2,2-trichloroacetic acid (TCA) precipitated to remove unincorporated 32P NAD+. The purified samples were loaded on SDS-PAGE gels, Commassie stained, dried, and subjected to autoradiography. Reactions analyzing activation of PARP1 by human SIRT6 contained 2 µl PARP1 (Sigma, P0996), 5 µg SIRT6WT ormutant proteins, 20 mM Tris$Cl (pH 8.0), 100 mM NaCl, 10 mM MgCl2, 10 µM ZnCl2, 10% glycerol, 300 µM NAD+, 1 mM DTT, and 0.1 µg/ml sonicated salmon sperm DNA in a 50-µl volume. These reactions were performed at 30°C for 30 min. The samples were then analyzed by western blotting using anti-PAR antibody (BD Pharmigen, 550781).
3
Immunolocalization of PAR and TbPARP
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Immunolocalization experiments were carried out as detailed in our previous work [33 (link)] with 1:500 polyclonal anti- PAR antibody (BD) made in rabbit to detect PAR and 1:100 polyclonal anti-TbPARP antibody (GenScript) made in rabbit to detect TbPARP. Nuclear and kinetoplast DNA were stained with DAPI (2 μg/mL) (Sigma).
4
Oxidative Stress Response in Trypanosoma
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Wild type bloodstream cultures were grown in HMI-11 medium for 24 h up to a density of 5 × 105 parasites/ml, and treated with 250 µM of H2O2 for 10 minutes in the same culture media. Transgenic procyclic cultures were grown in SDM-79 medium up to a density of 5 × 106 parasites/ml and treated with 500 µM of H2O2 for 10 minutes.
Parasites were fixed with 3.8% (W/V) formaldehyde in PBS at 4 °C, permeabilized with fresh PBS-0.1% Triton X-100 and blocked at room temperature for 1 h. PAR was detected with 1:500 rabbit polyclonal anti- PAR antibody (BD) followed by 1:500 Alexa Fluor 488 goat anti-rabbit IgG antibody (Invitrogen). TbPARP was detected with 1:100 rabbit polyclonal anti-TbPARP antibody (GenScript), followed by 1:500 Alexa Fluor 594 goat anti-rabbit IgG antibody (Invitrogen). Fusion proteins TBP-PARP-eYFP, TBP-N-eYFP and TBP-WRA-eYFP were localized with fluorescence of eYFP. Excess of antibody was removed by 3 × 5 min washes in PBS, and nuclear and kinetoplast DNA stained with 2 μg/ml DAPI (Sigma). Coverslips were washed with distilled water and mounted in Mowiol and then visualized using an Olympus BX41 microscope.
Parasites were fixed with 3.8% (W/V) formaldehyde in PBS at 4 °C, permeabilized with fresh PBS-0.1% Triton X-100 and blocked at room temperature for 1 h. PAR was detected with 1:500 rabbit polyclonal anti- PAR antibody (BD) followed by 1:500 Alexa Fluor 488 goat anti-rabbit IgG antibody (Invitrogen). TbPARP was detected with 1:100 rabbit polyclonal anti-TbPARP antibody (GenScript), followed by 1:500 Alexa Fluor 594 goat anti-rabbit IgG antibody (Invitrogen). Fusion proteins TBP-PARP-eYFP, TBP-N-eYFP and TBP-WRA-eYFP were localized with fluorescence of eYFP. Excess of antibody was removed by 3 × 5 min washes in PBS, and nuclear and kinetoplast DNA stained with 2 μg/ml DAPI (Sigma). Coverslips were washed with distilled water and mounted in Mowiol and then visualized using an Olympus BX41 microscope.
5
Oxidative Stress and Protein Detection
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Parasites were treated for 10 min with 1 mM H2O2. Western blot analysis was carried out as it is detailed in our previous work [17 (link)] with 1:5.000 polyclonal anti-PAR antibody (BD) made in rabbit. As a loading control we used α-tubulin antibody.
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