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Bioreagents ez run prestained rec protein ladder

Manufactured by Thermo Fisher Scientific

The Bioreagents EZ-run prestained rec protein ladder is a molecular weight standard used for estimating the molecular weights of proteins in SDS-PAGE. It contains a set of pre-stained recombinant proteins with known molecular weights that can be visualized during electrophoresis.

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3 protocols using bioreagents ez run prestained rec protein ladder

1

Recombinant Protein Cleavage Assay

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Recombinant cat S was generated as previously described11 (link), 16 (link) and diluted in 1× PBS, pH 7.4, 5 mM EDTA, 5 mM DTT and 5 mM cysteine chloride. Papain (Sigma-Aldrich) was diluted in 0.1 × PBS, 50 mM sodium acetate, pH 6.5, 5 mM EDTA, 5 mM DTT and 5 mM cysteine chloride. Crystalline Papain is more active and has a broader cleavage range than recombinant cat S. Different incubation times or concentrations were thus used as indicated. N-terminal peptides derived from the sequence of MrgprC11 were obtained from GenScript and diluted in PBS. The 28 amino acid N-terminal peptide MDPTISSHDTESTPLNETGHPNCTPILT was made by Peptide 2.0 (Chantilly, VA). The cysteine protease inhibitor E-64 was obtained from Sigma-Aldrich. Similar results were obtained at either 5 or 10 µM of this compound. Anti-Gaussia luciferase, catalog # E8023S from New England Biolabs was used at a dilution of 1:1500. Anti-phospho-PKC (pan) (βII Ser660) catalog #9371 from Cell Signaling Techology was used at a dilution of 1:1,000. HRP-labeled donkey anti rabbit, catalog # NB660-894 and anti-mouse antibodies catalog # NB7544 were from Thermo Fisher and used at 1:2,000 and 1:5,000 respectively. MW markers were Bioreagents EZ-run prestained rec protein ladder, cat # 3603500 from Thermo Fisher. All other reagents were purchased from Invitrogen unless otherwise noted.
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2

Western Blot Analysis of Brown Adipocytes

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Mature brown adipocytes were lysed with radioimmunoprecipitation assay buffer (RIPA buffer) (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, and 1% Triton X-100), containing 0.1% SDS, 0.01% protease-inhibitor, 0.01% phosphatase-inhibitor cocktail II, and 0.01% phosphatase-inhibitor cocktail III (all from Sigma-Aldrich). Protein concentrations were measured using a BCA Protein Assay Kit (Thermo Fisher Scientific), with BSA dilution series as the standard. Proteins were separated by SDS–PAGE, with Fisher BioReagents EZ-Run Prestained Rec Protein Ladder (Thermo Fisher Scientific), as the molecular weight marker and transferred to a polyvinylidene fluoride (PVDF) Immobilon-PSQ membrane, 0.2 μm (Merck Millipore). Unspecific binding sites were blocked with 5% BSA/TBS-T. The membranes were incubated with primary antibody solutions, Abcam (Cat. no. ab10983), 1:1,000. The membranes were washed three times (each 10 min), with 1× PBS before incubation with secondary HRP-conjugated antibody (Cat. no. 7074, 1:10,000; Cell Signaling Technology). β-actin (HRP-linked, Cat. no. sc-47778, 1:5,000; Santa Cruz Biotechnology) was used as loading control. Quantifications were performed using Image J software.
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3

Recombinant Protein Cleavage Assay

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Recombinant cat S was generated as previously described11 (link), 16 (link) and diluted in 1× PBS, pH 7.4, 5 mM EDTA, 5 mM DTT and 5 mM cysteine chloride. Papain (Sigma-Aldrich) was diluted in 0.1 × PBS, 50 mM sodium acetate, pH 6.5, 5 mM EDTA, 5 mM DTT and 5 mM cysteine chloride. Crystalline Papain is more active and has a broader cleavage range than recombinant cat S. Different incubation times or concentrations were thus used as indicated. N-terminal peptides derived from the sequence of MrgprC11 were obtained from GenScript and diluted in PBS. The 28 amino acid N-terminal peptide MDPTISSHDTESTPLNETGHPNCTPILT was made by Peptide 2.0 (Chantilly, VA). The cysteine protease inhibitor E-64 was obtained from Sigma-Aldrich. Similar results were obtained at either 5 or 10 µM of this compound. Anti-Gaussia luciferase, catalog # E8023S from New England Biolabs was used at a dilution of 1:1500. Anti-phospho-PKC (pan) (βII Ser660) catalog #9371 from Cell Signaling Techology was used at a dilution of 1:1,000. HRP-labeled donkey anti rabbit, catalog # NB660-894 and anti-mouse antibodies catalog # NB7544 were from Thermo Fisher and used at 1:2,000 and 1:5,000 respectively. MW markers were Bioreagents EZ-run prestained rec protein ladder, cat # 3603500 from Thermo Fisher. All other reagents were purchased from Invitrogen unless otherwise noted.
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