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.
Mcp257
Manufactured by Enzo Life Sciences
Sourced in United Kingdom, United States
The MCP257 is a multi-channel pipette designed for accurate and precise liquid handling. It features adjustable volume settings and can be used for a variety of laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Mcp196, by Enzo Life Sciences (2 mentions)
Mcp79, by Enzo Life Sciences (2 mentions)
Mb01015, by GenScript (1 mentions)
Hydrobond c, by Cytiva (1 mentions)
P45 110, by Enzo Life Sciences (1 mentions)
Mcp20, by Enzo Life Sciences (1 mentions)
Hyperfilm ecl, by GE Healthcare (1 mentions)
Versa doc 1000 imaging system, by Bio-Rad (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Immobilon p, by Merck Group (1 mentions)
3 protocols using mcp257
1
Quantifying Proteasome Subunits in Rat Brain
2
Immunoblot Analysis of Proteasome Subunits
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Immunoblot analyses of α and β-subunits of constitutive and immuno proteasomes, α-tubulin, free ubiquitin and polyubiquitinated proteins were performed as previously described21 (link),54 (link). Briefly, extracts were resolved by 18% (for free ubiquitin) or 12% (for polyubiquitinated conjugates and all other antigens) SDS-PAGE gel and transferred on Nitocellulose (Sigma-Aldrich, St. Louis, MO, USA, for polyubiquitinated conjugates) or Immobilon®-P (Merck Millipore, Darmstadt, Germany, for all other antigens) transfer membranes. Nitrocellulose membrane was boiled for 5 min to unmask poly-Ub antigens and so make more sensitive and quantitative the immunoblot. The membranes were then incubated in blocking buffer (5% BSA, 0.1%Tween-20 in 1 × PBS), followed by incubation with mAb anti-ubiquitin (P4D1, Santa Cruz Biotechnologies, Santa Cruz, CA, USA), anti α-tubulin (T5168, Sigma-Aldrich, St. Louis, MO, USA), anti-α3, -α4, -α5 (MCP196, MCP79, MCP257, Enzo Life Sciences, Farmingdale, NY, USA), anti-β1, -β2, -β5, -β1i -β2i, -β5i (gift of Prof. S. Ferrone, Harvard Medical School, Boston, MA, USA). Bound antibodies were visualized using the ECL technique and bands were detected with Hyperfilm™ ECL™ (GE Healthcare). Densitometric analysis was performed with a VersaDoc 1000 Imaging System and Quantity One software (Bio-Rad, Hercules, CA, USA).
3
Proteasomal Subunits Immunoblot Analysis
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University of Turin's Institutional Research Information System and Open Access Institutional Repository
Immunoblot analyses of proteasomal non-catalytic sub-units b3, b4, b5, and a6, were performed as previously described (Favole et al., 2012) . Briefly, 75 ug of proteins recovered after ultracentrifugation of muscle extracts were separated on a 12% SDS-PAGE gel, and transferred on aImmobilon®-P transfer membrane (Millipore). The membrane was then incubated in blocking buffer (5% BSA, 0.1%Tween-20 in 1 × PBS), followed by incubation with primary monoclonal antibodies (MCP196, MCP106, MCP79,MCP257, Enzo Lifesciences). Bound antibodies were visualized using the ECL technique.
Immunoblot analyses of proteasomal non-catalytic sub-units b3, b4, b5, and a6, were performed as previously described (Favole et al., 2012) . Briefly, 75 ug of proteins recovered after ultracentrifugation of muscle extracts were separated on a 12% SDS-PAGE gel, and transferred on aImmobilon®-P transfer membrane (Millipore). The membrane was then incubated in blocking buffer (5% BSA, 0.1%Tween-20 in 1 × PBS), followed by incubation with primary monoclonal antibodies (MCP196, MCP106, MCP79,MCP257, Enzo Lifesciences). Bound antibodies were visualized using the ECL technique.
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