Crude brain homogenate was diluted 4:5 with 5× sample buffer (Genscript MB01015), boiled for 5 minutes then stored at -20°C. Samples were loaded at 20μg/ml total protein on 10% SDS-polyacrylamide gel for protein separation, transferred to nitrocellulose membrane (Hydrobond-C, Amersham) and probed with either anti-totalCaMKIIα (Santa Cruz sc-136212, 1:10000) or anti-phospho-CaMKII (Santa Cruz sc-12886-R, 1:200) and the relevant secondary antibody (IRDye from LI-COR, anti-mouse for total CaMKIIα and anti-rabbit for phospho-CaMKII). Bands were detected using an Odyssey infrared fluorescent scanner, the integral of intensity quantified using Odyssey infrared imaging systems application software version 3.0.16 and expressed as ratios to rat cortex in arbitrary units.
Hydrobond c
Manufactured by Cytiva
Sourced in United Kingdom
Hydrobond-C is a laboratory equipment product designed for use in various scientific applications. It serves as a core component in various experimental setups, providing a reliable and consistent platform for conducting research and analysis. The product's primary function is to facilitate the controlled and precise handling of liquids, samples, and other materials within the laboratory environment.
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2 protocols using hydrobond c
1
CaMKII Expression and Phosphorylation Analysis
2
Quantifying Proteasome Subunits in Rat Brain
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Western blotting was undertaken as previously described [35 (link)]. Briefly, crude brain homogenate was diluted 4:5 with 5x sample buffer (Genscript MB01015, USA), boiled for 5 min then stored at –20°C. Samples were loaded at 20μg/ml total protein on 10% SDS-polyacrylamide gel for protein separation, transferred to nitrocellulose membrane (Hydrobond C, Amersham Biosciences, Amersham, UK), and probed with Proteasome 19S ATPase subunit RPT6 (p45-110, Enzo Life Sciences, Exeter, UK, 1:2000), Proteasome 20S α3 subunit (MCP257, Enzo Life Sciences, Exeter, UK, 1:2000) Proteasome 20S α6 subunit, (MCP20, Enzo Life Sciences, Exeter, UK, 1:2000). Bands were detected using an Odyssey infrared fluorescent scanner, the integral of intensity quantified using Odyssey infrared imaging systems application software version 3.0.16 and expressed as ratios to rat cortex run on the same gel in arbitrary units. All samples were run in duplicate.
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