Activation of the STAT3 pathway was assessed by quantifying pSTAT3tyr705 using immunoblot analysis with detection of fluorescent signals using an infrared fluorescence scanner (Licor Biosciences, Lincoln, NE) or with detection using an ECL chemiluminescent substrate (Amersham Biosciences, Piscataway, NJ) captured onto x-ray film (Fuji Medical systems, Stamford, CT) as described previously [12] (link), [19] (link). Briefly, following incubation with primary antibodies (anti-phospho-STAT3tyr705[1∶500]), blots were washed with phosphate buffered saline with 0.1% Tween 20 and incubated with fluorescent-labeled secondary antibodies (1∶2500) for 1 h prior to scanning by Licor or using a Personal Densitometer (Molecular Dynamics, Sunnyvale, CA).
Ecl chemiluminescent substrate
Manufactured by Cytiva
Sourced in United States
ECL chemiluminescent substrate is a reagent used in Western blotting techniques to detect and quantify target proteins. It provides a sensitive and reproducible method for visualizing protein bands on a membrane. The substrate undergoes a chemiluminescent reaction when exposed to the enzyme-labeled antibodies, producing light that can be captured and analyzed.
Automatically generated - may contain errors
3 protocols using ecl chemiluminescent substrate
1
Quantification of Activated STAT3 Pathway
2
Western Blot Protein Quantification
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Cells were seeded in six-well plates and allowed to grow up to 80% confluence. After the indicated treatments, total cell proteins were extracted and quantified. An equal amount of 40 μg of protein extract was loaded to each lane on a 12% SDS-PAGE gel. The proteins were then transferred to a nitrocellulose membrane (GE Healthcare, San Francisco, CA, USA). The membranes were later blocked in Tris-buffered saline (TBS) with 5% skim milk for 1 h and then incubated in the corresponding primary antibodies overnight. The membranes were then incubated with secondary antibodies at room temperature for 1 h. Immunoreactivity was detected using an ECL chemiluminescent substrate (Amersham Pharmacia Biotech, Piscataway, NJ, USA). GAPDH protein was synchronously detected as a loading control.
3
Protein Expression Analysis by Western Blot
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Protein expression was compared by Western blot using mouse polyclonal anti-AKR1C3 (Sigma Co.). Briefly, cells were seeded in 6-well plates and cultured to 70% confluence. After the indicated treatments, cell protein extracts were prepared. Western blots were performed with 40 μg of protein extract and protein was detected using ECL chemiluminescent substrate (Amersham Pharmacia Biotech, Piscataway, NJ). GAPDH protein was used as a loading control. Western blot experiments were repeated at least three times.
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