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).
Mcp196
Manufactured by Enzo Life Sciences
Sourced in United States
The MCP196 is a laboratory instrument designed for the measurement and analysis of protein concentration. It utilizes a spectrophotometric method to determine the amount of protein present in a sample. The core function of the MCP196 is to provide accurate and reliable protein concentration data to support various research and testing applications.
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Lab products found in correlation
2 protocols using mcp196
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Immunoblot Analysis of Proteasome Subunits
2
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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