Protease inhibitors complete edta free

Manufactured by Roche

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Protease inhibitors (complete EDTA-free) are a type of laboratory equipment used to inhibit the activity of proteases, which are enzymes that break down proteins. These inhibitors are designed to be used in various research and diagnostic applications that require the preservation of protein structure and function.

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Lab products found in correlation

Ibl 637, by PerkinElmer (1 mentions) Lysozyme, by Merck Group (1 mentions) Dnase 1, by Roche (1 mentions) Glutathione sepharose 4b resin, by GE Healthcare (1 mentions) Easytag express 35s, by PerkinElmer (1 mentions) Prism 7, by GraphPad (1 mentions) Typhoon scanner, by GE Healthcare (1 mentions) Nupage 4 to 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)

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Protease inhibitors (4287 citations) Complete protease inhibitor (1691 citations) Complete EDTA-free (281 citations) Complete EDTA-free protease inhibitor (261 citations) EDTA-free protease inhibitor (209 citations) Protease inhibitors (Complete, (20 citations) Protease Complete (7 citations) Complete EDTA-free protease (5 citations) Complete protease inhibitor-EDTA (3 citations) Complete EDTA-free Protease Inhibitors cocktail (3 citations)

5 protocols using protease inhibitors complete edta free

Radiation-Induced Protein Extraction

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Two days after reverse or standard transfection, the HaCaT cells were irradiated with 2 Gy from a ¹³⁷Cs source (IBL 637, CisBio International, Saclay, France) at a dose rate of 1.724 Gy/min or the Anémone/Bio irradiator (60Co, 2 Gy/min) in the ARC-Nucléart facility at CEA-Grenoble. After irradiation, the cultures were returned to the incubator. Thirty minutes later, the reverse-transfected cells were fixed in ice-cold methanol for 1 min and then washed three times with phosphate-buffered saline and stored at 4 °C. Standard transfected cells were lysed after 30 min of incubation using RIPA buffer (50 mM Tris–HCl, pH 7.4, 1 % Nonidet P-40, 0.25 % sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM Na₃VO₄, and 1 mM NaF) supplemented with 1× protease inhibitors (complete EDTA-free, Roche, Indianapolis, IN) to extract total proteins.

Benzina S., Pitaval A., Lemercier C., Lustremant C., Frouin V., Wu N., Papine A., Soussaline F., Romeo P.H, & Gidrol X. (2015). A kinome-targeted RNAi-based screen links FGF signaling to H2AX phosphorylation in response to radiation. Cellular and Molecular Life Sciences, 72(18), 3559-3573.

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Robust Cell Lysis and Clarification

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Pelleted cells were resuspended in PBS containing lysozyme (Sigma), DNase I (Roche Applied Science), and protease inhibitors (Complete EDTA-free; Roche Applied Science). Cells were then mechanically lysed using a single pass through a cell disruptor at 30 kp.s.i. (Constant Systems). Cell lysate was clarified by centrifugation at 30,000 × g for 30 min. The supernatant was passed through a 0.22-μm filter before further purification.

Dixon E.V., Claridge J.K., Harvey D.J., Baruah K., Yu X., Vesiljevic S., Mattick S., Pritchard L.K., Krishna B., Scanlan C.N., Schnell J.R., Higgins M.K., Zitzmann N, & Crispin M. (2014). Fragments of Bacterial Endoglycosidase S and Immunoglobulin G Reveal Subdomains of Each That Contribute to Deglycosylation. The Journal of Biological Chemistry, 289(20), 13876-13889.

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Recombinant Protein Expression and Purification

Cited 1 time

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Recombinant proteins were expressed in E. coli BL21(DE3) cells as GST-fusion proteins with a TEV protease cleavage site after the GST-tag. Cells were grown at 37°C to an OD₆₀₀ of 0.6 when protein expression was induced with 0.3 mM IPTG and the cultures were shifted to 25°C. Cells were harvested 20 h after induction. Selenomethionine substituted SidC (residues 1-608) for structure determination was expressed similarly according to (24 (link)). Cells were collected by centrifugation and resuspended in lysis buffer (20 mM Hepes pH 7.8, 300 mM NaCl, 1 mM PMSF, 0.5 mM TCEP) supplemented with protease inhibitors (complete EDTA-free, Roche) and lysed using a cell disruptor (Avestin).
Cells lysates were cleared by centrifugation and the GST-fusion proteins isolated by affinity chromatography using glutathione-sepharose 4B resin (GE Life Science). The proteins were eluted by on column cleavage from the GST-tag with His-TEV protease at 4°C overnight. The TEV protease was removed by subsequent Ni-NTA chromatography. SidC constructs for crystallization were further purified by size exclusion chromatography (Superdex S200 16/60) in 20 mM Hepes pH 7.8, 300 mM NaCl, 0.5 mM TCEP and concentrated to ~24 mg/ml.
MBP-RalF fusion proteins and His-tagged ΔN17Arf1 protein were purified as described in (19 (link)).

Horenkamp F.A., Mukherjee S., Alix E., Schauder C.M., Hubber A.M., Roy C.R, & Reinisch K.M. (2014). Legionella pneumophila subversion of host vesicular transport by SidC effector proteins. Traffic (Copenhagen, Denmark), 15(5), 488-499.

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Purification of Insoluble Brain Proteins

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Purification of the insoluble fraction in brain tissue samples was performed as previously described^{16 (link)}. In brief, tissue was homogenized in 1 ml tissue lysis buffer (10 % (w:v) sucrose, 10 mM HEPES pH 7.0, 800 mM NaCl, 5 mM Ethylenediaminetetraacetic acid (EDTA), 1 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA), 1 × protease inhibitors Complete EDTA-free (Roche), 1 × phosphatase inhibitors (Pierce)) with a small pistil, vigorously mixed (30 s), sonicated (30 s), and tissue debris removed by centrifugation (18,000 × g, 30 min, 4 °C). The cleared tissue lysate supernatant was brought to 1 % N-lauroylsarcosine (v:v), vigorously mixed (30 min, 24 °C), centrifuged (18,000 × g, 30 min, 4 °C). Protein aggregates were precipitated from the second supernatant by ultracentrifugation (100,000 × g, 1 h, 4 °C) and the protein pellet was isotope labeled and re-solubilized in one step (2 % formaldehyde, 0.3 M sodium cyanoborohydride, in 100 mM Hepes pH 7.0, 10 μl final volume, vigorously mixing, 15 min, 24 °C). The dimethylation reaction was quenched with ammonium bicarbonate (1% final w:v, 5 min, 24 °C). Proteins were denatured (8 M guanidinium chloride, 10 mM TCEP) for 1 h at 37 °C and free sulfhydryl groups alkylated (20 mM iodoacetamide, 30 min, 24 °C).

Bamberger C., Pankow S., Martínez-Bartolomé S., Ma M., Diedrich J., Rissman R.A, & Yates JR I.I.I. (2021). Protein Footprinting via Covalent Protein Painting Reveals Structural Changes of the Proteome in Alzheimer Disease. Journal of proteome research, 20(5), 2762-2771.

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Pulse-Chase Protein Trafficking Assay

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Cells were starved in Methionine/Cysteine-free DMEM (Gibco, #21013024) for 30 min, pulsed with 15 uCi/mL ³⁵Smethionine/cysteine (EasyTag EXPRESS ³⁵S, PerkinElmer) for 30 min and chased in complete DMEM containing 10% FBS. At each time point, media and cells were collected and cells were lysed in Buffer A (50 mM Tris, pH 7, 150 mM NaCl, 1% [vol/vol] Triton X-100, and 2 mM ethylenediaminetetracetic acid (EDTA)) supplemented with Protease inhibitors (Complete EDTA-free, Roche). Media and cell lysates were precleared, and the protein of interest was immunoprecipitated. Radiolabeled immunoprecipitated proteins were eluted in Loading buffer (50 mM Tris, pH 6.8, 0.1% [vol/vol] glycerol, 20% [p/v] SDS, 5% [vol/vol] b-mercaptoethanol, and 1 mg/mL Bromophenol Blue), separated on NuPAGE 4 to 12% Bis-Tris gels (Thermo Fisher Scientific), and detected by phosphorimaging using a Typhoon scanner (GE Healthcare). The protein bands were quantified using Fiji, and the percentage of the mature or secreted band in each sample was plotted with Prism 7.0 (GraphPad Software). For experiments with 4-PBA, compound was added to a final concentration of 10 mM during the starvation phase and included at the same concentration in the pulse and chase media.

Gomez-Navarro N., Maldutyte J., Poljak K., Peak-Chew S.Y., Orme J., Bisnett B.J., Lamb C.H., Boyce M., Gianni D, & Miller E.A. (2022). Selective inhibition of protein secretion by abrogating receptor–coat interactions during ER export. Proceedings of the National Academy of Sciences of the United States of America, 119(31), e2202080119.

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