To detect GFP NS and GFP-Ala6 ubiquitylation (Figures 1C , 4C and S5E ), GFP immunoprecipitation was performed under semi-denaturing conditions. HeLa cells were grown on 15 cm dishes and treated with MG132 for 4 h before harvesting in ice-cold lysis buffer (20 mM TRIS pH 7.5, 150 mM NaCl, 0.5% Triton, 1 mM EDTA, 100 mM NEM (N-Ethylmaleimide), supplemented with EDTA-free protease inhibitor tablet (Roche)). After clarification, lysates were incubated with 20 μl GFP trap magnetic agarose and rotated for 3 h at 4°C. Beads were washed 1x in 10 mM TRIS pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 100 mM NEM, supplemented with EDTA-free protease inhibitor tablet (Roche), 3x in stringent wash buffer (8 M UREA, 1% SDS in PBS), and 1x in 1% SDS/PBS. Proteins were eluted by boiling for 10 min at 95°C in 40 μl 2x Laemmli Buffer and analyzed by immunoblot.
Edta free protease inhibitor tablet
Manufactured by Roche
Sourced in Germany, Switzerland, Canada
EDTA-free protease inhibitor tablet is a laboratory reagent used to prevent the degradation of proteins during sample preparation and analysis. It inhibits the activity of proteases, enzymes that can break down proteins, without the use of EDTA, a common chelating agent. This product helps maintain the integrity of protein samples for downstream applications.
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Lab products found in correlation
Emulsiflex c3, by Avestin (4 mentions)
Histrap nickel column, by GE Healthcare (3 mentions)
Escherichia coli bl21 star de3 cells, by Thermo Fisher Scientific (3 mentions)
C3 emulsiflex, by Roche (2 mentions)
Histrap hp column, by GE Healthcare (2 mentions)
Superdex 75 26 60, by GE Healthcare (2 mentions)
400 mesh copper grid, by Electron Microscopy Sciences (2 mentions)
Porcine brain tubulin, by Cytoskeleton (2 mentions)
Paclitaxel, by Cytoskeleton (2 mentions)
Bugbuster, by Merck Group (2 mentions)
Black half area 3993 nonbinding surface 96 well plates, by Corning (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Phosphatase inhibitor tablet, by Roche (2 mentions)
Flag peptide, by Merck Group (2 mentions)
Benzonase, by Merck Group (2 mentions)
Ab6672, by Abcam (2 mentions)
Rala 610222 specific antibody, by BD (2 mentions)
Hdac1 h 51, by Santa Cruz Biotechnology (2 mentions)
Sb203580, by Selleck Chemicals (2 mentions)
Dmrie c reagent, by Thermo Fisher Scientific (2 mentions)
82 protocols using edta free protease inhibitor tablet
1
Detection of GFP Ubiquitylation
2
Purification of PTP1B Protein Variants
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Cell pellets were resuspended in ice-cold lysis buffer (50 mM Tris pH 8.0, 500 mM NaCl, 5 mM imidazole, 0.1% Triton X-100, and EDTA-free protease inhibitor tablets [Roche]) and lysed by high-pressure cell homogenization (Avestin C3 Emulsiflex). The bacterial lysate was clarified by centrifugation at 45,000 x g for 60 min at 4 °C. After filtration, the supernatant was loaded onto a HisTrap HP column (GE Healthcare) quilibrated with 50 mM Tris pH 7.5, 5 mM imidazole, and 500 mM NaCl, and the His6-tagged protein was eluted using a 5–500 mM imidazole gradient. Fractions containing PTP1B were pooled and cleaved with tobacco etch virus (TEV) protease overnight at 4 °C while being dialyzed against 50 mM Tris pH 8.0, 500 mM NaCl. Cleaved protein was further purified using Ni2+-NTA immobilized metal affinity chromatography followed by size exclusion chromatography (SEC; Superdex 75 26/60; GE Healthcare) equilibrated in NMR buffer (50 mM HEPES pH 6.8, 150 mM NaCl, 0.5 mM TCEP) to a purity of >98% and a final yield of 40 mg PTP1B1-301 or 20 mg PTP1B1-393 per liter of LB cell culture. Purified PTP1B1-301 and PTP1B1-393 were concentrated to 1 mM and 0.3 mM, respectively. For long-term storage, the protein was flash frozen in liquid nitrogen and stored at −80 °C.
3
Purification of Recombinant CRD-BP Protein
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The plasmid pET28b(+)-CRD-BP which contains the entire coding region of mouse CRD-BP cDNA was a generous gift from Dr. Jeffrey Ross, University of Wisconsin. The mouse CRD-BP cDNA in this plasmid is flanked with the FLAG tag epitope at its N-terminus and a 6xHis-tag at its C-terminus. Recombinant CRD-BP was purified from Escherichia coli BL21(DE3) cells using a 1 mL bed volume of nickel-NTA (QIAGEN) column under denaturing conditions. Proteins eluted from the column at either pH 5.4 or 4.5 were subjected to a series of dialysis steps (3 hours at each step) pH 5/6 M urea, pH 5.5/4 M urea, pH 6/2 M urea, pH 6.7/1M urea and pH 7.4/0 M urea in a buffer containing 200 mM KCl, 1 mM EDTA, 10% (v/v) glycerol, 1 mM reduced glutathione, 0.1 mM oxidized glutathione, 0.01% (v/v) Triton X-100, 20 mM triethanolamine [13] (link), and EDTA-free protease inhibitor tablets (Roche, Laval, Quebec). Following dialysis, the samples were centrifuged at 13,200 rpm for 30 minutes to remove any precipitated proteins. The purified protein solutions were then quantified using the Quick Start Bradford 1 x Dye Reagent (Bio-Rad, Mississauga, Ontario) and analyzed for purity using Coomasie blue-stained 12% SDS-PAGE.
4
Purification and Analysis of Microtubules
5
Purification and Characterization of Proteins
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All general chemicals were purchased from Fisher Scientific and Sigma. Plasmid constructs were synthesized by Genscript USA, Inc. (Piscataway, NJ, USA). BugBuster was purchased from EMD Millipore Corp. (Billerica, MA, USA). Column resins and PD-10 gel filtration columns were purchased from Cytiva Inc. (Marlborough, MA, USA). EDTA-free protease inhibitor tablets were from Roche, Inc. (Mannheim, Germany). Black half-area Corning #3993 nonbinding surface 96-well plates were from Corning Inc. (Corning, NY, USA). Tris-HEPES acrylamide gels (8–16% gradient) and SDS-PAGE running buffer were from Thermo Scientific (Rockford, IL, USA). The JCSG + screen was from Qiagen (Valencia, CA, USA). The gel imager is from BioRad Inc. (Hercules, CA, USA).
6
Purification of PTP1B Protein Variants
7
Quantifying Active Rac1 GTPase Levels
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Active Rac1 was affinity purified from lysates using an effector pull-down approach with GST-PAK beads. Cells were serum starved for 24 hours before treatment. At the relevant time, cells were lysed in ice-cold lysis buffer [20 mM Hepes pH 7.5, 140 mM NaCl, 1% (v/v) Igepal, 4 mM EDTA, 4 mM EGTA, 0.5% (wt/vol) sodium deoxycholate, 10% (vol/vol) glycerol] supplemented with EDTA-free protease inhibitor tablets (Roche). Lysates were clarified by centrifugation at 12,000 g, 4 °C for 5 minutes prior to snap-freezing in liquid nitrogen to preserve GTPase-activity while other batched samples were processed. Thawed lysates were then incubated with 20 µg GST-PAK beads (Cytoskeleton) for 1 hour at 4 °C. Beads were washed three times with ice-cold lysis buffer, and active Rac1 was eluted off beads by addition of Laemmli reducing sample buffer. For each condition, equal volumes of GTP-Rac1 eluted from the GST-PAK beads, and equal volume of ‘total’ extract obtained prior to snap-freezing were resolved by SDS-PAGE and analysed by Western blotting. The ratio between GTP-Rac1 and total Rac1 was quantified to determine the Rac1 activation state.
8
RIPK1 Immunoprecipitation and Western Blot
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BMDMs were cultured in 10 cm dishes and treated as indicated. Media was removed and plates washed with ice-cold PBS. Cells were lysed by scraping with cell lysis buffer containing 20 mM Tris-HCl, pH 7.5, 135 mM NaCl, 1.5 mM Mg2Cl, 1 mM EGTA, 1% Triton X-100 (Sigma-Aldrich), 10% glycerol, EDTA-free protease inhibitor tablets (Roche, Basel, Switzerland), and phosphatase inhibitor tablets (Roche). Cell lysates were rotated at 4 °C for 20 min and then clarified at 4 °C at 14,000 rpm for 15 min. Protein G agarose beads (25 μl) were blocked for 1 h with 1% BSA in lysis buffer and then bound with 1.5 μg mouse anti-RIPK1 antibody (38/RIP; BD Biosciences). RIPK1 was immunoprecipitated with rotation at 4 °C for 4 h. Beads were washed four times with lysis buffer and eluted by heating to 95 °C in 50 μl of 2X NuPAGE LDS sample buffer (Life Technologies) containing 5% β-mercaptoethanol. Eluted proteins were subjected to Western blot analysis.
9
TRIM14 Protein Purification Protocol
10
Purification of S. cerevisiae Atg Proteins
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If not stated otherwise S. cerevisiae Atg proteins were purified at 4°C. Pre-cooled lysis buffer containing 50 mM Tris HCl pH 8.3, 300 mM NaCl (180 mM NaCl for protein complexes), 5% glycerol, 2 mM DTT, EDTA free protease inhibitor tablets (Roche), 2 mM EDTA, 0.2 mM PMSF, 1 mM benzamidine and Pierce universal nuclease was added to bacterial or insect cell pellets. The lysis buffer used for bacterial protein purifications was supplemented with lysozyme (100 μg/ml). Protease inhibitor tablets and irreversible protease inhibitors were omitted for the purification of enzymes with an active site cysteine. Instead PMSF (0.2 mM) leupeptin (10 μM), pepstatin A (10 μM) and EDTA (4 mM) were used. Cells were lysed by sonication and spun at 20,000 rpm for one hour using a JA-20 rotor.
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