For Western blots, tissues or cells were lysed in the NP‐40 lysis buffer containing 20 mM Tris‐HCl (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Nonidet P‐40, 10 mM sodium fluoride, 1 mM sodium pyrophosphate, 1 mM sodium orthovanadate, and Cømplete protease inhibitor (Roche Applied Science). Lysates containing 20 or 40 μg of protein were separated by SDS/PAGE and transferred to Immobilon P membranes (Millipore, Billerica, MA, USA). Membranes were blocked with 5% nonfat dry milk (in Tris‐buffered saline, 0.05% Tween‐20) for 1 h at room temperature. They were then probed with the primary antibodies for at 4°C 8 h. After incubation with horseradish peroxidase‐conjugated secondary antibodies (anti‐mouse IgG, Pierce, Rockford, IL; anti‐rabbit IgG, GE Healthcare Amersham, Buckinghamshire, UK), bound proteins were detected by incubation with Immobilon chemiluminescent substrate (Millipore).
C mplete protease inhibitor
Manufactured by Roche
Cømplete protease inhibitor is a laboratory reagent designed to inhibit a wide range of protease enzymes. It is used to prevent the degradation of proteins during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using c mplete protease inhibitor
1
Western Blot Protein Detection Protocol
2
Soluble Protein Extraction from Brain Regions
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The hippocampus, entorhinal cortex, and temporal cortex specimens were flash frozen in liquid nitrogen immediately upon resection and stored in polyethylene screw-cap tubes below −70 °C. Each specimen was homogenized at the same tissue concentration in order to improve comparisons. Brain pieces were dissected (100–200 mg, avoiding blood and white matter as much as possible) and pulverized on dry ice, transferred to pre-weighed polyethylene tubes on dry ice, weighed, and immediately sonicated in 5 vol. ice cold buffer H (100 mM potassium phosphate (pH 7.4), 10 mM ethylenediaminetetraacetic acid (EDTA), 1 mM dithiothreitol containing Cømplete® Protease Inhibitor (Roche)), then centrifuged at 15K×g for 20 min at 4 °C. A small portion of each supernatant was assayed for total protein (microplate Bradford assay), and the remainder of the soluble protein extracts split into several aliquots that were stored frozen below −70 °C.
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