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.
P45 110
Manufactured by Enzo Life Sciences
Sourced in United Kingdom
The P45-110 is a laboratory equipment product offered by Enzo Life Sciences. It is a device designed for a specific core function within a laboratory setting. However, a detailed and unbiased description of its exact capabilities and features cannot be provided without the risk of extrapolation or interpretation. Therefore, the description for this product is not available.
Automatically generated - may contain errors
Lab products found in correlation
Mb01015, by GenScript (1 mentions)
Hydrobond c, by Cytiva (1 mentions)
Mcp257, by Enzo Life Sciences (1 mentions)
Mcp20, by Enzo Life Sciences (1 mentions)
Malachite green, by Merck Group (1 mentions)
Picrotoxin, by Bio-Techne (1 mentions)
Mcp231, by Enzo Life Sciences (1 mentions)
Adenosine triphosphate (atp), by Merck Group (1 mentions)
Complete protease inhibitor, by Roche (1 mentions)
2 protocols using p45 110
1
Quantifying Proteasome Subunits in Rat Brain
2
Proteasome Subunit Immunodetection Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Anti-20S core α subunits monoclonal mouse antibody (mAb MCP231) and Rpt6 regulatory subunit monoclonal mouse antibody (mAb p45-110) were purchased from Enzo Life Sciences. Rabbit PSD-95 antibody was purchased from Calbiochem. Custom rabbit (pAb; clone #07) anti-Rpt6 phospho-specific antibody for S120 (pS120) was previously generated commercially (ProSci) against a synthetic phosphorylated peptide (Djakovic et al., 2012 (link)). Fluorescent and HRP-conjugated secondary antibodies were purchased from Thermo Fisher Scientific. N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC) substrate was used (BACHEM). GST-Ubl and His-UIM constructs were a gift from the Alfred Goldberg lab. Malachite Green and Adenosine triphosphate were purchased from Sigma-Aldrich. PhosSTOP kinase/phosphatase inhibitor and cOmplete protease inhibitors were purchased from Roche. Picrotoxin was purchased from Tocris.
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