Human mesothelial MeT-5A cells were seeded to confluency in two wells of a six well plate and allowed to adhere overnight. One well was treated with 10 µL of MCAA+ serum (1:200 dilution) and the other well was treated with 10 µL IgG-cleared MCAA+ serum. After 2h, a nuclear extraction (Signosis, Santa Clara, CA) was performed according to the manufacturer’s protocol and samples were stored at 4°C. To screen the nuclear extracts for 48 different transcription factors, TF Activation Profiling Plate Array I (Signosis) was utilized and performed according to the provided protocol. Briefly, nuclear extracts were mixed with biotin-labeled probes for the consensus sequences of specific transcription factor binding sites. Bound probes were then separated using a spin column. The bound probes were separated from the transcription factor complex and analyzed on the hybridization plate, which is coated with complementary sequences of the probes. The plate was then developed, with luminescence detected and quantified on the microtiter plate reader.
Tf activation profiling plate array 1
Manufactured by Signosis
Sourced in United States
The TF Activation Profiling Plate Array I is a lab equipment product that enables the assessment of transcription factor (TF) activation profiles. It provides a platform for the simultaneous measurement of multiple TF activities in a single experiment.
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Lab products found in correlation
5 protocols using tf activation profiling plate array 1
1
Transcription Factor Activation Profiling
2
Transcription Factor Activation in Malaria
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RAW-ELAM cells (3 × 106 cells/well) were added to 6-well plates and incubated for 12 to 16 h. CS2-WT or CS2-SBP1-KO iRBCs (15 × 106 cells/well) were added and incubated for 4 h. Cells were washed to remove iRBCs, and nuclear extracts were prepared by using a nuclear extraction kit (Signosis) according to the manufacturer's instructions. The activation levels of 48 TFs were measured by using TF Activation Profiling Plate Array I (Signosis). Briefly, biotin-labeled probes encoding TF DNA-binding-site consensus sequences were incubated with 8 μg of nuclear extract. Active TFs bound their respective probes, and unbound probes were washed away. Subsequently, bound probes were eluted and hybridized to complementary sequences on a 96-well plate. Luminescence was measured on a Chameleon V plate reader. Values were normalized to the TFIID value, and the fold change of CS2-SBP1-KO over CS2-WT was calculated. A ≥2-fold change between CS2-WT and CS2-SBP1-KO was considered a difference. Independent experiments were repeated three times on separate days and with freshly isolated iRBCs.
3
Transcription Factor Activation Profiling after C3 Treatment
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For screening the transcriptional activity of 48 different transcription factors after treatment with C3 for 48 h, the TF Activation Profiling Plate Array I (Signosis Inc., Santa Clara, CA, USA) was performed. HT22 cells were incubated with 500 nM C3 or medium for control conditions. After 48 h, the nuclear extraction (Signosis Inc., Santa Clara, CA, USA) was performed according to manufacturers’ instructions. The protein concentration was determined by Bradford assay, and 5 μg of nuclear extracts per condition were applied in TF Activation Profiling Plate Array I according to manufacturers’ instructions. Both conditions were measured on one 96-well plate containing two sets for each 48 transcription factors. The luminescence was detected at Synergy4 microplate reader (BioTek Instruments Inc., Winooski, VT, USA). For each condition, the relative light units of the transcription factors were normalized to the value of the non-regulated SATB1 as internal control. The relative regulation was calculated by the ratio of C3-treatment in comparison to control condition. Significant regulations were estimated in a twofold increase or decrease of transcriptional activity. Transcription factors whose activity was altered in all three experiments significantly in the same direction were defined as regulated.
4
Transcription Factor Activation Profiling
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Transcription factor activity assays are performed according to TF Activation Profiling Plate Array I kit instructions (Signosis). Nuclear extract/probe mixture (15 µg nuclear extract and 3 µl biotin-labeled probe mix I) incubated at RT for 30 min. Free probes were isolated from TF/probe complexes using a spin column purification procedure, although individual probes can locate their target TF and form complexes with it. The hybridization plate was used to separate the bound probes from the complex for analysis. The captured DNA probes were further identified with Streptavidin-HRP Conjugate and then assessed with a chemiluminescent plate reader (Veritas microplate luminometer). On a microplate luminometer, luminescence is measured in relative light units (RLUs).
5
Probing SLNCR1 Interactions via MS2 Pulldown
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