Full-length Casp6 was expressed at 30 °C in E. coli BL21(DE3)pLysS (Promega, Fitchburg, WI, USA) and purified using Ni Sepharose Fast Flow 6 (GE Healthcare, Little Chalfont, UK)59 (link) using Tris buffers pH 8.0. Eluted Casp6 was diluted fivefold with buffer A1 (20 mM Tris pH 8.5, 2 mM DTT, 5% glycerol), loaded on Macro Prep High Q resin (Bio-Rad Laboratories, Hercules, CA, USA), washed with 20 mM Tris pH 8.5, 2 mM DTT, 5% glycerol, 50 mM NaCl and eluted with 80-260 mM NaCl gradient in buffer A1. Casp6 large and small subunits were co-expressed at 23 °C in E. coli BL21(DE3)pLysS and purified as described using Tris buffers pH 8.5. Casp6-C163A was purified as in59 (link) and Casp3 as in7 (link). Caspase purity was assessed by SDS-PAGE and Coomassie blue staining. Protein concentration was measured using Quick Start Bradford 1× Dye Reagent (Bio-Rad Laboratories).
Macro prep high q resin
Manufactured by Bio-Rad
Sourced in United States
Macro-Prep High Q resin is a strong anion exchange chromatography media designed for the purification of proteins, peptides, and other biomolecules. It features a macroporous structure and a high level of cross-linking for improved mechanical and chemical stability. The resin's large pore size allows for efficient mass transfer and high dynamic binding capacity.
Automatically generated - may contain errors
Lab products found in correlation
Ni sepharose 6 fast flow, by GE Healthcare (2 mentions)
Bl21 de3 plyss, by Promega (1 mentions)
Quick start bradford 1 dye reagent, by Bio-Rad (1 mentions)
Bl21 de3 plyss competent cells, by Promega (1 mentions)
E coli bl21 de3 cells, by Promega (1 mentions)
Ktaprime plus, by GE Healthcare (1 mentions)
Heparin sepharose column, by GE Healthcare (1 mentions)
Uno q1 column, by Bio-Rad (1 mentions)
4 protocols using macro prep high q resin
1
Casp6 Expression and Purification
2
Purification of Casp6 Recombinant Protein
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The pET23b( +)-Casp6WT-His or pET23b( +)-Casp6N73T-His plasmids were expressed in BL21(DE3)pLysS competent cells (Promega, Madison, WI, USA). Casp6 was purified using Ni Sepharose Fast Flow 6 (GE Healthcare, Chicago, IL, USA) and Macro Prep High Q Resin (Bio-Rad Laboratories, Hercules, CA, USA) as described32 (link),46 (link). Pure protein was aliquoted, fast frozen in EtOH/dry ice bath, and stored at − 80 °C freezer.
3
Purification and Kinetic Analysis of GES-5 Enzyme
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For protein expression, competent E. coli BL21(DE3) cells (PROMEGA, Madison, WI, USA) were transformed with recombinant plasmid blaGES-5/pet24a(+) as reported [20 (link)]. Protein purification was performed using an Äkta Prime plus chromatograph system (GE Healthcare Life Sciences, Freiburg, Germany). GES-5 (Uniprot ID Q09HD0) was conveniently purified in a single step using a Macro-Prep High Q resin (Bio-Rad, Hercules, CA, USA.). For the obtained protein, the KM values for the two reporter substrates, CENTA and nitrocefin were determined and found to be 524 μM and 208 μM, respectively [27 ].
4
Purification of Porcine RNA Polymerase II and Human U1 snRNP
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The porcine Pol II was purified from S. scrofa thymus as described (27) . In brief, the thymus was flash frozen in liquid nitrogen and homogenized in a blender. After removing the fat tissue, the supernatant was precipitated with 0.04% polyethylenimine (PEI). The PEI pellet was dissolved in 0.4 M ammonium sulfate buffer and applied onto a column packed with Macro-Prep High Q resin (Biorad). The eluted peak was then precipitated with 4 M ammonium sulfate and the re-dissolved pellet applied onto an 8WG16 antibody column. The eluate was further purified on a Uno Q1 column (Biorad) with a final size exclusion purification step using the Sephacryl S-300 16/60 column (GE Healthcare) in 10 mM HEPES pH 7.25, 150 mM NaCl, 10 µM ZnCl2 and 10 mM DTT. Human U1 snRNP was purified from HeLa cell nuclear extract by immunoaffinity purification and a 10-30% glycerol gradient as described (28) . The fractions containing U1 snRNP were diluted to 100 mM NaCl and applied onto a heparin sepharose column (GE Healthcare). U1 snRNP was eluted with 20 mM Tris-HCl pH 7.9, 450 mM NaCl, 1.5 mM MgCl2 and 1 mM DTT.
Human P-TEFb, PAF, DSIF and SPT6 were purified as described (27) .
Human P-TEFb, PAF, DSIF and SPT6 were purified as described (27) .
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