Panc-1 cells were seeded onto 15 cm diameter plates. At a confluence of 80%, the cells were treated with 1 and 0.5 mM H2O2 in serum-free DMEM High Glucose medium for 30 min. Then the nuclear proteins were extracted and quantified as described in the “Nuclear extract and biotin-streptavidin pull down assay”. For immunoprecipitation, 1.5 mg of Protein A-Dynabeads (ThermoFisher Scientific-Invitrogen, Waltham, MA, USA) were incubated with 3 μg of PAR antibody (Poly/Mono-ADP Ribose (E6F6A) Rabbit mAb #83732, Cell Signalling Technology, Leiden, The Netherlands) and 3 μg of IgG Rabbit (ThermoFisher Scientific-Invitrogen, Waltham, MA, USA) as negative control in 20 mM Tris-HCl, pH 7.4, 150 mM KCl, 8% glycerol, 1mM DTT and 0.1 mM ZnAc for 15 min at RT. After one wash with the same buffer, 80 μg of nuclear extracts were allowed to react with anti-PAR- and IgG Rabbit-derivatized Dynabeads for 30 min at RT. The beads were captured with a magnet and washed twice with the same buffer. The proteins were denatured and eluted with Laemmli buffer (4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.004% bromophenol blue and 0.125 M Tris–HCl).
Igg rabbit
Manufactured by Thermo Fisher Scientific
Sourced in Italy
The IgG Rabbit is a laboratory equipment product designed for use in scientific research. It serves as an immunoglobulin G (IgG) antibody, derived from rabbit serum. The core function of this product is to provide a reliable and consistent source of IgG antibodies for various immunological applications.
Automatically generated - may contain errors
Lab products found in correlation
Magnify chromatin immunoprecipitation system, by Thermo Fisher Scientific (2 mentions)
Anti gli1 h300, by Santa Cruz Biotechnology (1 mentions)
Anti acetyl histone 3, by Merck Group (1 mentions)
H3k4me2, by Abcam (1 mentions)
Microson xl 2000, by Bioventus (1 mentions)
H3k9me3, by Abcam (1 mentions)
4 protocols using igg rabbit
1
Oxidative Stress-Induced PAR Signaling
2
Oxidative Stress-Induced Nuclear Protein Interactions
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Panc-1
cells were seeded
onto 15 cm diameter plates. At 80% confluence, the cells were treated
with 0.1 mM H2O2 in serum-free DMEM high-glucose
medium for 30 min. Then the nuclear proteins were extracted and quantified
as described in theNuclear Extract and Biotin–Streptavidin
Pull-Down Assay section. For immunoprecipitation, 1.5 mg of
Protein A-Dynabeads (ThermoFisher Scientific-Invitrogen, Waltham,
MA, USA) was incubated with 3 μg of anti-PAR (Poly/Mono-ADP
Ribose (E6F6A) Rabbit mAb #83732, Cell Signaling Technology, Leiden,
The Netherlands), anti-PARP-1 (46D11, Cell Signaling Technology, Leiden,
The Netherlands), anti-Ku70 (D10A7, Cell Signaling Technology, Leiden,
The Netherlands), anti-MAZ (clone 133.7, IgG mouse, Santa Cruz Biotechnology,
Dallas, TX, USA), anti-hnRNPA1 (clone 9H10, IgG mouse, Merck Life
Science, Milano, Italy), and IgG Rabbit (ThermoFisher Scientific-Invitrogen,
Waltham, MA, USA) as negative control in 20 mM Tris–HCl (pH
7.4), 20 mM KCl, 8% glycerol, 1 mM DTT, and 0.1 mM ZnAc for 15 min
at RT. After one wash with the same buffer, 80 μg was allowed
to react with anti-PAR- and IgG Rabbit-derivatized Dynabeads for 30
min at RT. The beads were captured with a magnet and washed three
times with the same buffer. The proteins were denatured and eluted
with Laemmli buffer (4% SDS, 20% glycerol, 10% 2-mercaptoethanol,
0.004% bromophenol blue, and 0.125 M Tris–HCl).
cells were seeded
onto 15 cm diameter plates. At 80% confluence, the cells were treated
with 0.1 mM H2O2 in serum-free DMEM high-glucose
medium for 30 min. Then the nuclear proteins were extracted and quantified
as described in the
Pull-Down Assay
Protein A-Dynabeads (ThermoFisher Scientific-Invitrogen, Waltham,
MA, USA) was incubated with 3 μg of anti-PAR (Poly/Mono-ADP
Ribose (E6F6A) Rabbit mAb #83732, Cell Signaling Technology, Leiden,
The Netherlands), anti-PARP-1 (46D11, Cell Signaling Technology, Leiden,
The Netherlands), anti-Ku70 (D10A7, Cell Signaling Technology, Leiden,
The Netherlands), anti-MAZ (clone 133.7, IgG mouse, Santa Cruz Biotechnology,
Dallas, TX, USA), anti-hnRNPA1 (clone 9H10, IgG mouse, Merck Life
Science, Milano, Italy), and IgG Rabbit (ThermoFisher Scientific-Invitrogen,
Waltham, MA, USA) as negative control in 20 mM Tris–HCl (pH
7.4), 20 mM KCl, 8% glycerol, 1 mM DTT, and 0.1 mM ZnAc for 15 min
at RT. After one wash with the same buffer, 80 μg was allowed
to react with anti-PAR- and IgG Rabbit-derivatized Dynabeads for 30
min at RT. The beads were captured with a magnet and washed three
times with the same buffer. The proteins were denatured and eluted
with Laemmli buffer (4% SDS, 20% glycerol, 10% 2-mercaptoethanol,
0.004% bromophenol blue, and 0.125 M Tris–HCl).
3
Chromatin Immunoprecipitation of Gli1 and Acetyl-H3
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ChIP was performed using the MAGnify Chromatin Immunoprecipitation System (Invitrogen). Protocol was performed as described in44 (link). For each ChIP reaction 300,000 cells were used, cell lysates were added to their respective antibody/beads for 2 h. Eluted DNA was PCR amplified with primers encompassing the Gli- responsive elements of human ABC promoter. The following antibodies were used: IgG rabbit (Invitrogen), rabbit polyclonal anti-Gli1 H300 (sc-20687, Santa Cruz Biotechnology Inc.), rabbit polyclonal anti-acetyl-histone 3 (06599, Millipore). Eluted DNA has been analysed with Q-PCR. Primers were designed with Primer-Blast designing tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/ ) and Primers tool (Genomatix Genome Analyzer, GGA, v3.30126, https://www.genomatix.de/ ) and are reported in Supplementary Table 2 .
4
ChIP Assay with MAGnify System
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ChIP assays with the MAGnify Chromatin Immunoprecipitation System (Invitrogen) were performed according to manufacturer's protocol. Confluent cells were crosslinked in 1% formaldehyde for 10 minutes, sheared to 100–700 base pair fragments with a Misonix Microson XL 2000 at power level 10 repeating a 5 seconds on/5 seconds off cycle 30–35 times on ice. Invitrogen Dynabeads were incubated with the following antibodies for 1–2 hours at 4°C: 1 μL IgG rabbit (Invitrogen), 2 μL UBF (Santa Cruz 9131), 4–5 μL TRF2 (Novus NB110-57130), 2 μL H3K4me2 (Abcam ab7766), and 1.5 μL H3K9me3 (Abcam ab8898). 150,000 cells were used in each immunoprecipitation (IP) and incubated with the antibody/Dynabeads for 2–3 hours at 4°C. Beads were washed, chromatin reverse crosslinked, and DNA eluted according to the MAGnify ChIP protocol. ChIPs were repeated at least 3 times for each modification.
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