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Sk 0001

Manufactured by Signosis
Sourced in United States

SK-0001 is a precision laboratory instrument designed for accurate measurement and analysis. It features advanced sensor technology and digital display for reliable data collection. The core function of SK-0001 is to provide precise quantitative information for scientific research and testing applications.

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3 protocols using sk 0001

1

Analyzing EMT-related Protein Expression

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Following incubation, cells were washed with PBS three times and lysed in 250-μL lysis buffer (5-mM bicine buffer, 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF 0.3 mM), leupeptin (10 μg/mL), and aprotonin (2 μg/mL)). Supernatants were obtained after centrifuging (8000 rpm) the cell lysates. Nuclear proteins were extracted to detect Snail using an extraction kit (SK-0001; Signosis, Santa Clara, CA, USA). Cells were collected and rinsed with ice-cold PBS, incubated in working reagent I for 10 min, and then centrifuged (8000 rpm) for 5 min at 4 °C. The supernatant was then collected and stored. The pellet was incubated in working reagent II for 2 h and centrifuged (8000 rpm) for 5 min. The supernatant was then collected for Snail detection. Protein concentrations were determined by the Bradford protein assay (Bio-Rad, Hercules, CA, USA). The total proteins from cell lysates (25 μg) and nuclear proteins (25 μg) were electrophoresed in a 12.5% SDS-PAGE gel. After protein transfer, the PVDF membranes were blocked with 5% dehydrated skim milk and probed with primary antibodies (anti-human Akt, E-cadherin, FAK, MMP-2, MMP-9, N-cadherin, PI3K, Snail, TIMP-1, TIMP-2, mTOR, uPA, p-PI3K, p-Akt, p-mTOR, and actin antibodies) at 4 °C for 16 h, followed by secondary antibodies for 2 h. ECL reagent was used to detect protein expressions.
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2

Cytokine-Induced Transcription Factor Activation

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BMDMs were treated with different cytokines. Nuclear proteins were isolated with a nuclear extraction kit (Signosis, SK-0001), and analyzed by using a 96-well plate transcription factor (TF) activation array (Signosis, FA1002) according to the manufacturer’s instruction. The activity of each transcriptional factor was normalized as the fold of TFIID’s activity.
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3

Profiling Transcription Factor Activation in Cancer Stem Cells

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To simultaneously measure the activities of multiple TFs, a Cancer Stem Cell TF activation profiling plate array (FA-1004; Signosis, Santa Clara, CA, USA) was used. Briefly, biotin-labeled probes containing consensus sequences of TF DNA-binding sites were incubated with nuclear extracts that were prepared by nuclear extraction kit (SK-0001; Signosis) for 30 min at 25°C. The TF/probe complex mixtures were separated by spin column purification. The bound probes were detached from the complex using elution buffer and centrifuged at 9,800 ×g for 2 min. After the eluents were denatured at 98°C for 5 min, the denatured sample was added to TF hybridization buffer. The resulting mixture (100 μL) was added to each well of the hybridization plate, and the plate was sealed with aluminum adhesive and incubated at 42°C for 16 h. The captured DNA probe was further detected using a streptavidin-horseradish peroxidase conjugate. Endpoint luminescence readings of the samples were observed using Fluostar omega (BMG Labtech, Ortenberg, Germany).
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