Restriction enzymes, Phusion Hot Start DNA polymerase, and Quick Ligation Kit were from New England Biolabs (Ipswich, MA). Bacto Tryptone and Bacto Yeast extract were from BD (Franklin Lakes, NJ). HP ampicillin was from Geno Technology (Maryland Height, MO). HisPur Cobalt chromatography cartridges were from Pierce Biotechnology (Rockford, IL). HiTrap SP HP and HiTrap Q HP ion exchange chromatography cartridges were from GE Healthcare (Piscataway, NJ). His-Tag monoclonal antibody was from Novagen by EMD Millipore (Darmstadt, Germany). BCA protein assay kit was from Pierce Biotechnology (Rockford, IL). Calbiochem Protease Inhibitor Cocktail II was from EMD Millipore (Darmstadt, Germany). [3H] cAMP was from Moravek Biochemicals (Brea, CA). [α-32P] ATP was from Perkin Elmer (Boston, MA). All other chemicals and reagents used were from Sigma-Aldrich (St. Louis, MO).
Calbiochem protease inhibitor cocktail 2
Manufactured by Merck Group
Sourced in Germany
Calbiochem® protease inhibitor cocktail II is a ready-to-use solution designed to inhibit a broad spectrum of proteases. It is suitable for use in cell and tissue lysates to prevent protein degradation during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Quick ligation kit, by New England Biolabs (1 mentions)
Restriction enzyme, by New England Biolabs (1 mentions)
Bacto yeast extract, by BD (1 mentions)
Bacto tryptone, by BD (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
α 32p atp, by PerkinElmer (1 mentions)
Phusion hot start dna polymerase, by New England Biolabs (1 mentions)
Hitrap sp hp, by GE Healthcare (1 mentions)
Hitrap, by Cytiva (1 mentions)
Tissuelyzer, by Qiagen (1 mentions)
Halt, by Thermo Fisher Scientific (1 mentions)
Tissuelyser, by Qiagen (1 mentions)
Protease inhibitor, by Roche (1 mentions)
3 protocols using calbiochem protease inhibitor cocktail 2
1
Purification and Characterization of Recombinant Proteins
2
Tissue Lysate Enzymatic Assays
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For enzymatic assays, lysates of colonic and hepatic tissues were prepared in Tris buffer (100 mM Tris, 300 mM KCl, 0.1 % Triton X-100, pH 7.0, Calbiochem® protease inhibitor cocktail II (Merck Millipore, Darmstadt, Germany)) using a TissueLyzer (2 × 30 sec; 30 Hz; Qiagen, Hilden, Germany), centrifuged (14.000 x g, 30 min, 4°C) and stored at -80°C until further analysis. Total GPx and total TrxR activities were determined as previously described [47 (link)]. GPx4 activities were measured according to the measurement of total GPx activity, but by applying the specific substrate phosphatidylcholine hydroperoxide (PCOOH, 1.25 mM) instead of 0.00375 % H2O2 [24 (link)].
3
Hepatic Enzyme Activity Profiling Protocol
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For analysis of hepatic GPx, TR, NADPH quinone dehydrogenase 1 (NQO1), and GST activity liver tissue lysates were prepared in Tris buffer (100 mM Tris, 300 mM KCl, 0.1 % Triton X-100, pH 7.6; Calbiochem® protease inhibitor cocktail II (Merck Millipore, Darmstadt Germany)) using a tissue lyser (Qiagen). Lysates for the determination of hepatic superoxide dismutase (SOD) activity were prepared in 0.05 M potassium phosphate buffer containing protease inhibitor (ROCHE, Basel Switzerland). For Western blot analyses tissue lysates were prepared in RIPA buffer (50 mM Tris, 150 mM NaCl 2 , 0.5 % DOC, 1 % NP-40, 0.1 % SDS) containing protease and phosphatase inhibitors (HALT TM , Thermo Scientific). All lysates were centrifuged for 30 min at 14.000 x g and 4°C. Supernatants were stored at -80 °C until further analyses.
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