Mixed glial cultures were washed with PBS, and lysates were prepared by incubation in ice-cold RIPA buffer (50 mM Tris pH 7.4, 150 mM NaCL, 1% triton, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA) supplemented with protease inhibitors. Lysates were cleared by centrifugation, and protein concentration was measured by Bradford assay. Lysates were mixed with SDS sample buffer, boiled, and resolved by SDS-PAGE. Bands were transferred to PVDF Immobilon P membranes (Millipore) using a wet transfer, blocked in TBS-T containing 5% milk, and probed overnight at 4°C with primary antibody against arginase −1. Membranes were then incubated for 1 hour with HRP-conjugated secondary antibody (Jackson ImmunoResearch). Immunoblots were visualized using the SuperSignal West ECL system (Thermo Scientific) followed by film exposure. Blots were then stripped using Restore Western blot stripping buffer (Thermo Scientific, catalog # 21059), blocked again as above, and probed overnight at 4°C with primary antibody against actin, prior to visualization as above. Quantification was performed using ImageJ software.
Supersignal west ecl system
Manufactured by Thermo Fisher Scientific
The SuperSignal West ECL system is a chemiluminescent detection reagent used for western blotting applications. It provides a sensitive method for detecting and quantifying proteins that have been separated by gel electrophoresis and transferred to a membrane.
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2 protocols using supersignal west ecl system
1
Quantification of Arginase-1 Expression
2
Arginase-1 Quantification in Mixed Glial Cultures
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Mixed glial cultures were washed with PBS, and lysates were prepared by incubation in ice-cold RIPA buffer (50 mM Tris pH 7.4, 150 mM NaCL, 1% triton, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA) supplemented with protease inhibitors. Lysates were cleared by centrifugation, and protein concentration was measured by Bradford assay. Lysates were mixed with SDS sample buffer, boiled, and resolved by SDS-PAGE. Bands were transferred to PVDF Immobilon P membranes (Millipore) using a wet transfer, blocked in TBS-T containing 5% milk, and probed overnight at 4°C with primary antibody against arginase -1. Membranes were then incubated for 1 hour with HRP-conjugated secondary antibody (Jackson ImmunoResearch). Immunoblots were visualized using the SuperSignal West ECL system (Thermo Scientific) followed by film exposure. Blots were then stripped using Restore Western blot stripping buffer (Thermo Scientific, catalog # 21059), blocked again as above, and probed overnight at 4°C with primary antibody against actin, prior to visualization as above. Quantification was performed using ImageJ software.
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