Mitochondrial pellets (15 mg) from HeLa-NDUFA10FLAG and HEK293T-NDUFA10E160A/R161A cells were lysed at 1 mg/mL in lysis buffer B (50 mM Tris-HCl pH 7.4; 150 mM NaCl; 1% TritonTM X-100; 0.5% sodium deoxycholate) supplemented with 1× cOmplete™ protease inhibitor cocktail without EDTA (Roche), and 1× phosphatase inhibitor cocktail PhosSTOP™ (Roche). The lysates were precleared with 20 µL/mg of Mouse IgG Agarose (Sigma-Aldrich # A0919) for 1 h at 4 °C in an orbital shaker to reduce non-specific binding. After centrifugation at 1,000 × g for 5 min at 4 °C, the beads were discarded and the precleared lysate was incubated with ANTI-FLAG M2 Magnetic Beads (30 µL/mg) (Sigma-Aldrich # M8823) for 2 h at 4 °C in an orbital shaker. ANTI-FLAG M2 Magnetic Beads were washed 5 × 10 mL and 1 × 1 mL with lysis buffer B supplemented with 1x cOmplete™ protease inhibitor cocktail without EDTA (Roche). Finally, protein was eluted with 1 beads-volume of a 3xFLAG peptide at 150 ng/µL in TBS buffer (50 mM Tris-HCl pH 7.4; 150 mM NaCl) supplemented with 1x cOmplete™ protease inhibitor cocktail without EDTA (Roche). Eluted proteins and beads were stored at –80 °C until further experiments, and immunoprecipitation (IP) efficiency was monitored by western blot. See Supplementary Table 3 for a complete list of immunoprecipitation reagents and tools used in the study.
Complete protease inhibitor cocktail without edta
Manufactured by Roche
Sourced in Germany
The Complete protease inhibitor cocktail without EDTA is a laboratory product designed to provide a comprehensive solution for inhibiting a wide range of proteases. It is formulated to be used without the presence of EDTA, which can interfere with certain downstream applications. The product's core function is to effectively inhibit proteolytic activity in various experimental settings, helping to preserve the integrity of target proteins during sample preparation and analysis.
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Lab products found in correlation
Phosstop, by Roche (2 mentions)
Complete protease inhibitor cocktail, by Roche (2 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Excel 2013, by GraphPad (1 mentions)
Complex 1 immunocapture kit, by Abcam (1 mentions)
Phosstop phosphatase inhibitor cocktail, by Roche (1 mentions)
Anti flag m2 antibody ab, by Merck Group (1 mentions)
Ni nta affinity chromatography, by Qiagen (1 mentions)
Mono q, by GE Healthcare (1 mentions)
N ethylmaleimide, by Merck Group (1 mentions)
X tremegene hp, by Roche (1 mentions)
Mouse igg agarose, by Merck Group (1 mentions)
Anti flag m2 magnetic bead, by Merck Group (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Ecl prime western blotting detection kit, by GE Healthcare (1 mentions)
12 protocols using complete protease inhibitor cocktail without edta
1
Affinity Purification of FLAG-tagged Proteins
2
Chromogenic 4-NPF Substrate Assay
3
Mitochondrial DNA-Binding Assay
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Mouse liver mitochondria were lysed for 15 min at 4 °C in 100 mM phosphate buffer (KH2PO4), 1% DDM, 1× cOmplete™ protease inhibitor cocktail without EDTA (Roche), and 1× phosphatase inhibitor cocktail PhosSTOP™ (Roche). After centrifugation at 20,000 × g for 20 min at 4 °C, 10 µg of the mitochondrial extract were incubated with 0.06 pmol of [α-32P]-dGTP (3000 Ci/mmol) in binding buffer (75 mM Hepes pH 7.9; 50% glycerol; 180 mM NaCl; 2 mM DTT; 2 mM phenylmethylsulfonyl fluoride, and 2 µg/µL BSA) for 30 min at 30 °C. Cold competitors were added at 100-molar excess 30 min prior to [α-32P]-dGTP addition. Samples were run in native 6% acrylamide:bisacrylamide (37.5:1) gels. After electrophoresis, gels were vacuum-dried on a filter paper and developed by autoradiography.
4
Affinity Purification of Complex I
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Mitochondrial pellets (5 mg) from mouse liver were lysed at 5 mg/mL in DDM buffer for 30 min on ice. The lysate was centrifuged at 25,000 × g for 30 min at 4 °C (INPUT). The resulting supernatant was then divided and incubated in an orbital shaker overnight at 4 °C with pre-equilibrated Complex I Immunocapture Kit (Abcam #ab109711) beads (150 µg antibody/mg) or the equivalent amount of Immobilized Protein G Agarose beads (Abcam #ab174816) as a blank control. Next day, we separated FLOW-THROUGH aliquots from both incubations and washed the beads 1 × 8 mL and 1 × 1 mL with washing buffer (PBS; 0.05 % DDM; 1× cOmplete™ protease inhibitor cocktail without EDTA [Roche]; 1x phosphatase inhibitor cocktail PhosSTOP™ [Roche]). Finally, a 1/15 fraction of beads (ELUATES) was separated for western-blot monitoring of pulled-down proteins, and the remaining beads were processed for TCA-dNTP extraction.
5
Purification and Labeling of HIF-PHs and VBC Complex
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Recombinant human HIF-PHs were purified from Sf9 insect cell lysates. After infection with the recombinant virus stocks, generated via cloning of the respective cDNAs into pBacPAC transfer vectors (Clontech Laboratories), Sf9 cells were incubated for 48 h at 27°C with continuous shaking. Expression of the recombinant protein was confirmed by SDS-PAGE and western blot analysis of the expressed proteins. The lysate was centrifuged at 75,000 g at 4°C for 30 min and the supernatant containing the soluble HIF-PHs was incubated for 1 h at 2–8°C with CM Sepharose. After several wash steps, bound proteins were eluted from Sepharose using a linear NaCl gradient. Fractions containing HIF-PH activity were pooled, dialyzed against 100 mM Tris, 1.5 mM MgCl2, 2 mM DTT, 0.01% Tween-20, supplemented with 1% BSA and complete protease inhibitor cocktail without EDTA (Roche Diagnostics), and stored at –80°C. The VBC complex was expressed in Escherichia coli BL21(DE3) pLysS cells using the plasmid pST39-HisTrxNVHL-elongin B-elongin C. Protein expression and purification were performed as described previously [31] (link). DELFIA labeling reagent (PerkinElmer) was used to label purified VBC complex with europium according to the manufacturer’s instructions.
6
Protein Extraction and Immunoblot Analysis
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Cells were lysed in high salt lysis buffer (20 mM Tris-HCl pH 8.0, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, complete protease inhibitor cocktail without EDTA (Roche)) for 30 min on ice. After centrifugation (17,400 × g) at 4°C for 10 min, the supernatant was collected as whole cell lysate. Nuclear extracts were prepared with NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific). Immunoblot analysis was performed with anti-FLAG M2 antibody (Ab) (Sigma-Aldrich), anti-Pax5 Ab (sc-1974, Santa Cruz Biotechnology), anti-C/EBPβ Ab (sc-150, Santa Cruz Biotechnology), anti-β-tubulin Ab (MMS-410P, Covance), anti-MYH9 Ab (sc-47199, Santa Cruz Biotechnology), and anti-chicken Thy28 (cThy28) Ab (kindly gifted by Dr. Compton) [26 (link)].
7
scFv Bacterial Expression and Purification
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The scFv was PCR amplified from the pIT2 vector as a pelB-scFv construct and subcloned into a pet21d bacterial expression vector with or without integrated bacterial alkaline phosphatase29 (link) upstream of a C-terminal hexa-histidine tag by the NheI and NotI restriction sites. Protein expression was driven by the T7 promoter with IPTG inducible lac operon in BL21 E. coli host cells. The 2YT, ampicillin, 2% glucose o/n culture of a well isolated colony was inoculated 1:100 in 1 L 2 × TY, ampicillin, 0.1% glucose expression culture and incubated at 37 °C until OD600nm of 0.6–0.8. Protein expression was induced by 0.1 mM IPTG for 4–5 h at 25 °C, and pelleted cells were incubated for 2 h on ice in 50 ml 1 × TES buffer (0.2 M Tris-HCl pH 8, 0.5 mM EDTA, 0.5 M sucrose) supplemented with Complete Protease Inhibitor Cocktail without EDTA (Roche). Cells were pelleted and the supernatant was filtered (0.2 μm) and dialyzed twice against PBS using a 10 kDA cut-off snake skin dialyzing tube. The scFv or scFv-AP fusion protein was purified by Ni-NTA affinity chromatography (Qiagen) and anion exchange (Mono Q, GE healthcare). The purified proteins were quantified by spectroscopy using a Nanodrop 1000.
8
Subcellular Fractionation of C4-2 Cells
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5 × 106 C4–2 cells were collected, washed twice with cold PBS and lysed in cold hypotonic buffer (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT) supplemented with complete protease inhibitor cocktail without EDTA (Roche) on ice for 15 min. NP-40 was added to a final concentration of 0.625% and cells were vortexed vigorously for 10 s. Samples were centrifuged for 30 s at 16000 g and the supernatants were harvested as cytoplasmic fraction. The nuclear pellets were then resuspended in cold hypertonic buffer (20 mM HEPES pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT) supplemented with complete protease inhibitor cocktail (Roche). The samples were incubated at 4 °C for 15 min with agitation. The supernatants were collected after centrifugation for 5 min at 16000 g as nuclear proteins. Cytoplasmic and nuclear proteins were frozen in 5x sample buffer at − 20 °C until use.
9
Chromatin Immunoprecipitation and qPCR in Yeast
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Yeast cells (ca. 2 × 108 cells) were cultured and fixed with 1% formaldehyde. After quenching with 125 mM glycine, yeast cells were collected and lysed in 800 µl of Lysis buffer [0.1% sodium deoxycholate, 1 mM ethylenediaminetetraacetic acid (EDTA), 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-KOH, pH7.5, 140 mM NaCl, 1% Triton X-100, and cOmplete protease inhibitor cocktail without EDTA (Roche, Basel, Switzerland)] containing 0.5 mm glass beads, and crushed with a Bead-Beater. Chromatin DNA in supernatants was fragmented by sonication with a Smurt NR-50M (Microtec, Chiba, Japan) using the following parameters: 25% power output, 10 cycles of 30 s ON, and 60 s OFF, on ice. After centrifugation at 12,000 rpm for 15 min, a part of the supernatant (a volume equivalent to 4 × 106 cells) was subjected to enChIP followed by quantitative PCR (qPCR) analysis (enChIP-qPCR). enChIP-qPCR was performed as described previously [1 (link)]. Primers used in this study were as follows: gRNA_CAN1Y_SC_Y12-F_28924 (5′-tcagcgttctgtacttctccttc-3′) and gRNA_CAN1Y_SC_Y12-R_28925 (5′-aattgtatccattgcgctcttt-3′) for the CAN1 locus, gRNA_ADE2Z_SC_Y12-F_28928 (5′-aggaacatcaacatgctcaatct-3′) and gRNA_ADE2Z_SC_Y12-R_28929 (5′-aaataagcaactccaatgaccac-3′) for the ADE2 locus.
10
Ubiquitin-tagged Protein Purification
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U2OS cells (70% confluent, 10 cm dish) were transfected with 6xHis-tagged ubiquitin (15 μg) and FLAG-tagged histone H1.2 (5 μg) constructs using X-treme gene HP (Roche), one day before cell lysis according to manufacturer’s protocol. One hour before lysis, the cells were treated with 20 J/m2 UV. Cells were washed in PBS and harvested by scraping in 750 µl denaturing urea buffer (8 M urea, 300 mM NaCl, 50 mM Na2HPO4, 0.5% NP-40; pH 8.0) supplemented with 10 µM MG132 (Biomol), 10 mM N-ethylmaleimide (Sigma) and complete protease inhibitor cocktail without EDTA (Roche). Lysates were sonicated 3 times for 10 sec with amplitude 12 and centrifuged at 13,000 g and 4 °C for 15 min to remove remaining cell debris. Meanwhile 50 µl of Co2+ Sepharose beads slurry was equilibrated 3 times with urea buffer. Cleared lysates were incubated with Co2+ Sepharose beads for 2 h at 4 °C. Subsequently, beads were washed 4 times with urea buffer for 5 min and centrifuged at 3000 rpm for 1 min. His-tagged proteins were eluted by 20 min incubation with urea buffer containing 500 mM EDTA. Eluents were mixed with Laemmli buffer and separated on a Precast BioRad gel 5–14% and transferred to a PVDF membrane (0.45 µm).
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