Inhibition of APE1 redox activity was measured using an EMSA as routinely used in our laboratory and previously described [19 (link), 21 (link)]. Briefly, purified APE1 protein (10 mg/mL) was reduced with DTT (1.0 mM) at 37 °C for 10 min and then diluted with PBS buffer to final concentrations of APE1 and DTT of 2 mg/mL and 0.2 mM, respectively. Reduced APE1 protein and 6 μg of oxidized nuclear extracts (Hey-C2 cells, treated with 0.01 mM diamide for 10 min) were added, incubated for 30 min, and one mL of poly(dI-dC) · poly(dI-dC) (1 mg/L, Amersham Biosciences, Piscataway, NJ) was added for 5 min followed by one mL of the 5′ hexachlorofluorescein phosphoramidite (HEX)-labeled double-stranded oligonucleotide DNA (0.1 pmol, The Midland Certified Reagent Company, Midland, TX) containing the AP-1 consensus sequence (5′-CGCTTGATGACTCAGCCGGAA-3′). The mixture was further incubated for 30 min at room temperature. The final concentration of DTT in the redox reactions was 0.02 mM. Samples were analyzed using nondenaturing polyacrylamide gel and detected using the Hitachi FMBio II Fluorescence Imaging System (Hitachi Genetic Systems, South San Francisco, CA).
Poly di dc poly di dc
Manufactured by Cytiva
Sourced in United Kingdom
Poly(dI-dC) · poly(dI-dC) is a synthetic double-stranded DNA molecule commonly used as a non-specific competitor in various molecular biology applications. It serves as a control to help assess the specificity of DNA-protein interactions.
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Lab products found in correlation
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
γ 32p atp, by Cytiva (1 mentions)
Foetal calf serum, by Thermo Fisher Scientific (1 mentions)
Donkey anti goat igg horseradish peroxidase hrp, by Santa Cruz Biotechnology (1 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (1 mentions)
Lymphoprep, by Axis-Shield (1 mentions)
32p datp, by Cytiva (1 mentions)
T4 polynucleotide kinase, by Promega (1 mentions)
Nf κb gel shift oligonucleotide, by Promega (1 mentions)
Ap 1 gel shift oligonucleotide, by Promega (1 mentions)
3 protocols using poly di dc poly di dc
1
APE1 Redox Activity Measurement
2
NF-κB Activation Assay in THP-1 and J774 Cells
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Unless stated otherwise all materials were obtained from Sigma Aldrich (Poole, Dorset, U.K.) in their highest purity. RPMI, Dulbecco's modified Eagle medium (DMEM), phosphate buffered saline (PBS), foetal calf serum (FCS), Glutamax, penicillin and streptomycin were obtained from Invitrogen (Paisley, U.K.). THP-1 and J774 cells were obtained from the European Collection of Animal Cell Cultures (Salisbury, Wiltshire, U.K.). [γ-32P]-ATP (370MBq/ml, 10 mCi/ml), polyvinylidene difluoride membrane (PVDF) and poly(dI–dC)–poly(dI–dC) was obtained from Amersham Biosciences (Little Chalfont, Buckinghamshire, U.K.). NF-κB Gel Shift Assay System was obtained from Promega (Southampton, Hampshire, U.K.). Goat polyclonal antibodies for p50 (cross reactive with p105), rabbit polyclonal antibodies to p50, p65 or IκBα and actin, donkey anti-goat IgG–horseradish peroxidase (HRP) and supershift antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.). Donkey anti-goat IgG–fluorescein isothiocyanate (FITC) was obtained from AbD Serotec (Oxford, U.K.). Lymphoprep was obtained from Axis-Shield (Oslo, Norway).
3
Electrophoretic Mobility Shift Assay for NF-κB and AP-1
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NF-κB gel-shift oligonucleotide (5′-AGTTGAGGGGACTTTCCCAGGC-3′) and AP-1 gel-shift oligonucleotide (5′-CGCTTGATAGTCAGCCGGAA-3′; Promega Corp, Madison, WI, USA) were labeled with [32P]-dATP (Amersham Bioscience, Piscataway, NJ, USA), using T4 polynucleotide kinase (Promega Corp). End-labeled probe was purified from unincorporated [32P]-dATP using a purification column (Amersham Biosciences, Buckinghamshire, UK) and recovered in Tris-EDTA buffer (TE). Nuclear extracts (5 μg) were preincubated in buffer containing 12% glycerol, 12 mM HEPES, pH 7.9, 4 mM Tris-HCl, pH 7.9, 1 mM EDTA, 1 mM DTT, 25 mM KCl, 5 mM MgCl2, 0.04 μg/mL Poly (dI-dC) · Poly (dI-dC) (Amersham Bioscience), 0.4 mM PMSF, and TE. The labeled probe was added and the samples were incubated for 30 min at room temperature. The samples were subjected to electrophoretic separation at 4°C on a nondenaturing 5% acrylamide gel. The gel was dried at 80°C for 40 min and exposed to a radiography film for 6–18 h at −80°C with intensifying screens.
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