The DNA binding activity of transcription factors STAT3, NF-κB, Nrf-2, and AP-1 family was determined by DNA binding ELISA according to the manufacturer’s instructions (STAT3: Cat# 45196, NF-κB: Cat# 40096, Nrf-2: Cat# 50296; AP-1 family: Cat# 44296 TransAM® Kit, ActiveMotif, Rixensart, Belgium). Neuroblastoma cells were seeded in 100 mm dishes and incubated until sufficient colonization was achieved. The cells were washed with ice-cold PBS, scraped from the dish surface, centrifuged, and incubated with a hypotonic buffer. Nonidet P-40 was added and the cell lysis was checked under a microscope. After centrifugation and removal of the supernatant, a lysis buffer was added. The protein content of the lysates was determined by the Bradford method (Bradford, 1976). Samples containing 10 µg protein were then incubated with the primary antibodies for 1 hour, and the DNA binding activity of the transcription factors was analyzed by measuring the optical density at 450 nm on a microplate reader (Tecan®, Männedorf, Switzerland).
Transam kit
Manufactured by Active Motif
Sourced in United States, Belgium
The TransAM kit is a tool used to detect and quantify the activation of transcription factors in cell and tissue samples. It provides a sensitive and specific method for measuring the DNA-binding activity of transcription factors without the need for radioactivity or Western blotting.
Automatically generated - may contain errors
Lab products found in correlation
Nuclear extract kit, by Active Motif (3 mentions)
Nupage 10 bis tris gel, by Thermo Fisher Scientific (2 mentions)
Nuclear extraction kit, by Active Motif (2 mentions)
Microplate reader, by Tecan (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Simplechip enzymatic chromatin ip kit, by Cell Signaling Technology (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
Ecl plus detection kit, by Cytiva (1 mentions)
Nupage mops sds running buffer, by Thermo Fisher Scientific (1 mentions)
Trans blot semi dry system, by Bio-Rad (1 mentions)
Tris glycine buffer, by Bio-Rad (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Mouse anti β actin, by Merck Group (1 mentions)
Gen5 microplate collection analysis software data, by Agilent Technologies (1 mentions)
Polyvinylidene fluoride membrane, by Cytiva (1 mentions)
Bca protein assay reagent, by Thermo Fisher Scientific (1 mentions)
Nupage 4 12 bis tris mini gel, by Thermo Fisher Scientific (1 mentions)
Anti β actin ab, by Cell Signaling Technology (1 mentions)
Mpo activity, by Abcam (1 mentions)
Caspase 1 activity, by Abcam (1 mentions)
30 protocols using transam kit
1
Transcription Factor DNA Binding Assay
2
Western Blot Analysis of Protein Expression
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Whole-cell extracts were prepared with a nuclear extraction TransAm Kit (Active Motif, Carlsbad, CA, USA) and Western blotting was carried out as previously described.21 (link),22 (link) Briefly, samples were subjected to Western blot analysis (30–40 μg) in a NuPAGE 10% Bis-Tris Gel (Thermo Fisher Scientific). Blots were blocked with 5% nonfat dry milk in Tris-buffered saline, 0.1% Tween 20 for 1 hour at room temperature (RT), and then the blots were treated with a 1:200 dilution of primary antibody for 2 hours at RT. Blots were then incubated with secondary antibody (1:3,000 dilution) for 1 hour at RT and washed seven times with Tris-buffered saline, 0.1% Tween 20. Immunoreactive bands were visualized using an ECL Plus Detection Kit (Amersham, Pharmacia Biotech, Arlington Heights, IL, USA). Membranes were stripped and reprobed for β-actin, which was used as a protein loading control.
3
Quantification of c-Fos Binding to AP-1
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4h post-activation, splenocytes were collected and nuclear protein was isolated using a commercially available kit (Active Motif, Carlsbad, CA). Nuclear protein was then quantified by Bradford Assay (Bio-Rad, Hercules, CA). 5 μg of nuclear protein was used to determine binding of c-fos to the AP-1 consensus binding site, as determined by quantification with an ELISA-based assay (TransAM kit, Active Motif, Carlsbad, CA).
4
Measuring NF-κB DNA Binding Activity
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TransAM kit (Active Motif, Carlsbad, CA, USA) was used to estimate the DNA-binding activity of NF-κB in the nuclear extract according to the manufacturer’s instructions as described previously [32 (link)].
5
Quantifying Nrf2 Binding to ARE
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Nuclear lysates were prepared from cell lines using nuclear extract kit (Active Motif Inc., Carlsbad, CA, USA). Protein concentration was estimated using Bradford method. Nrf2 binding to ARE was determined using TransAM Kit based on manufacturer’s instructions (Active Motif Inc., Carlsbad, CA, USA). Six micro gram of protein were added per well of 96 well plate to which the oligonucleotide has been immobilized. The spectrophotometric read out was taken at 450nm and represented as binding activity of Nrf2.
6
Quantifying NF-κB Activity in Colon Tissues
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Nuclear extracts were prepared from colon tissues. Nuclear extracts was obtained with the use of a nuclear extraction kit (Active Motif). Binding activity of nuclear extract (5 μg) to NF-κB subunit (p50, p65, c-Rel) was measured using a Transam kit, obtained from Active Motif, as previously described 43 (link).
7
Quantifying Nrf2 DNA Binding Activity
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The Nrf2 activation was assessed using an ELISA kit (TransAM® Kit, Active Motif) according to manufacturer’s instructions. An ELISA-based assay consisting of an immobilized oligonucleotide containing the ARE consensus-binding site (5′-GTCACAGTG ACTCAGCAGAATCTG-3′) was used to measure Nrf2 DNA binding activity. Nrf2 from 20?μg of nuclear extract was allowed to bind to the ARE on 96-well plates. A primary antibody against Nrf2 was then used to detect bound Nrf2. A secondary antibody conjugated to HRP provided a colorimetric readout at 450?nm with reference length at 655 nm (GENios, TECAN®). Results were normalized to protein concentration in each sample. Values are expressed as fold increase of each group compared to the sham group.
8
ChIP-qPCR and Luciferase Assay for CHOP-Mediated Regulation of Tbx21
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ChIP assays were performed using the SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology). Chromatin was prepared from 4 × 106 wild-type or Ddit3−/− T cells that were activated for 72 h. Sheared chromatin was immunoprecipitated with antibodies against Chop, Histone 3 (D2B12 Rabbit antibody, #4260, Cell Signaling Technology), or rabbit IgG isotype (DA1E, #39600, Cell Signaling Technology). Purified DNA was analyzed by quantitative PCR with pre-validated primers that detect the Chop-binding sequence in the Tbx21 promoter (#GPM1043970(-)01A, SABiosciences-Qiagen). For DNA-binding activity, nucleic protein extracts were isolated from activated control and Ddit3 null T cells and monitored for binding of T-bet to a consensus DNA sequence through a specific TransAM® Kit (#51396, Active-Motif). For the dual luciferase gene reporter assays, plasmids harboring the Tbx21-CRE-SV-40 firefly luciferase and the SV-40 promoter-driven Renilla luciferase were co-transfected into 293T cells with pMSCV-Ddit3-IRES-Thy1.1 or control pMSCV- IRES-Thy1.1 vectors. After 48 h, dual luciferase assay was performed by Dual-Luciferase® Reporter Assay System (#E1910, Promega).
9
Quantifying NRF2 Binding in MDSC
10
Western Blot Analysis of Protein Expression
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Whole-cell extracts were prepared with a nuclear extraction TransAM kit (Active Motif, Carlsbad, CA, USA). Nuclear extracts (25–40 µg per lane) were separated on a NuPAGE 10% Bis-Tris Gel (Thermo Fisher Scientific). Electrophoresis was performed at 100 V for 2 hours using NuPAGE® MOPS SDS Running Buffer (Thermo Fisher Scientific). Separated proteins were electrotransferred at 25 V for 25 minutes to a polyvinylidene difluoride membrane using the Trans-Blot semi-dry system (Bio-Rad, Hercules, CA, USA) in Tris/glycine buffer (Bio-Rad). Blots were blocked overnight at 4°C in 5% nonfat dry milk in Tris-buffered saline (TBS) containing 0.075% Tween-20 (TBS-T), after which they were incubated with mouse anti-PR (AB-52) (1:200 dilution; Santa Cruz Biotechnology Inc., Dallas, TX, USA) or mouse anti-β-actin (1:2000; EMD Millipore) for 2 hours at room temperature. Blots were then washed three times with TBS-T for 10 minutes each, incubated with antimouse IgG HRP-linked antibody (Cell Signaling Technology, Danvers, MA, USA) for 1 hour at room temperature, and washed seven more times with TBS-T for 10 minutes each. Immunoreactive bands were visualized using the ECL plus detection kit (Amersham Pharmacia Biotech, Arlington Heights, IL, USA) using the Bio-Rad ChemiDoc XRS system.
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