Edta free protease inhibitor

Manufactured by Thermo Fisher Scientific

Sourced in United Kingdom, United States

EDTA-free protease inhibitors are chemical compounds designed to prevent the degradation of proteins by inhibiting the activity of proteases, which are enzymes that break down proteins. These inhibitors do not contain EDTA, a common chelating agent. They are typically used in laboratory settings to preserve the integrity of protein samples during experimental procedures.

Automatically generated - may contain errors

Lab products found in correlation

Female balb cj mice, by Janvier Labs (1 mentions) Pen strep, by Merck Group (1 mentions) Flag m2, by Merck Group (1 mentions) Irdye 800cw goat anti rabbit igg, by LI COR (1 mentions) Glass bead, by Merck Group (1 mentions) Odyssey fc imaging system, by LI COR (1 mentions) Fastprep, by MP Biomedicals (1 mentions) Simplyblue safestain, by Thermo Fisher Scientific (1 mentions) Glutathione sepharose 4b, by GE Healthcare (1 mentions) Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions) Malachite green assay kit, by Echelon Biosciences (1 mentions) Purelink genomic dna mini kit, by Thermo Fisher Scientific (1 mentions) Direct zol rna kit, by Zymo Research (1 mentions) Dynabeads protein g, by Thermo Fisher Scientific (1 mentions) Qubit rna br assay kit, by Thermo Fisher Scientific (1 mentions) Qubit rna hs assay kit, by Thermo Fisher Scientific (1 mentions) Isogen, by Nippon Gene (1 mentions) Syn per reagent protocol, by Thermo Fisher Scientific (1 mentions) Phosphatase inhibitor, by Roche (1 mentions) Image studio lite, by LI COR (1 mentions)

Spelling variants of this product

Protease inhibitors (1135 citations) EDTA-free protease and phosphatase inhibitors (2 citations) COmplete mini EDTA-free protease inhibitors (2 citations)

10 protocols using edta free protease inhibitor

Intranasal Infection of BALB/cJ Mice

Check if the same lab product or an alternative is used in the 5 most similar protocols

Female BALB/cJ mice were purchased from Janvier (Le Genest-Saint-Isle, France).
Inocula of WT and mutant bacteria used in this study are 2.10⁷ bacteria for KR WT, 2.10⁷, 4.10⁸ and 10⁹ for KR cps^- and 10⁹ for Kp52Δwzc. When appropriate, similar inocula of the respective bioluminescent strains were used.
Bacterial counts were determined as colony forming units (CFU) by plating serial dilutions of lung homogenates in 3 ml ice-cold PBS supplemented with 0,5% Triton X-100 and EDTA-free protease inhibitors (Fisher Scientific).
For survival studies, mice received either 2.10⁷ KR WT or 2.10⁷, 4.10⁸, 10⁹ KR cps^- by the intranasal route. Following infection, animals were returned to standard housing and observed for 14 days. A census of survivors was taken daily.

Corelli B., Almeida A.S., Sonego F., Castiglia V., Fevre C., Brisse S., Sansonetti P.J, & Tournebize R. (2018). Rhinoscleroma pathogenesis: The type K3 capsule of Klebsiella rhinoscleromatis is a virulence factor not involved in Mikulicz cells formation. PLoS Neglected Tropical Diseases, 12(1), e0006201.

+ Open protocol

+ Expand

Pulmonary Cytokine Profiling Post-Infection

Check if the same lab product or an alternative is used in the 5 most similar protocols

At various time post-infection the five pulmonary lobes were removed and collected in ceramic-beads containing tubes (Precellys lysing kit CK28) with 2,5 ml of ice cold PBS supplemented with 0,5% Triton X-100 and EDTA-free protease inhibitors (Fisher Scientific). Samples were then crushed using the Precellys homogenizer with the following program: 3 cycles of 15 sec at 5.000 × g with 10 sec pause. Twenty microliters were removed to determine the number of CFU/lung. After adding 10 μl of Pen/Strep (100X, Sigma), samples were centrifuged at 300 × g for 10 min and left on ice for 30 min. The supernatants were frozen rapidly in dry-ice ethanol bath and stored at -80°C. The following cytokines were measured: IL1ß, IL-10, IL-17, TNFα (Duoset, all from R&D Systems). Assays were performed according to the manufacturer’s instructions.

+ Open protocol

+ Expand

Protein Expression Analysis via SDS-PAGE

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

For protein expression analysis, strains were grown in selective media to an A₆₀₀ of 0.6 then harvested by centrifugation at 5000 x g. Cells were resuspended in lysis buffer (30 mM HEPES pH 7.4, 100 mM KCl, 0.1 mM MgCl₂, 10% glycerol + EDTA-free protease inhibitors (Thermo Fisher Scientific, Loughborough, United Kingdom). 200 μl of acid washed glass beads (Sigma-Aldrich, Poole, United Kingdom were added and cells were lysed by using a FastPrep (MP Bio, Santa Ana, CA) for 3 × 20 s at 6.5 ms⁻¹ with cooling on iced water for 5 min between cycles. Cell debris was removed from the cell extract by centrifugation at 10,000 × g for 15 min at 4°C. SDS-PAGE and immuno-blotting were performed as previously described (Jennings and Pavitt, 2010a (link)) using specific antibodies for eIF2α, eIF2γ, eIF2Bε, eIF5, eIF2Bγ, Flag-M2 (Sigma-Aldrich, Poole, United Kingdom), PAB1 (Encor Biotechnology, Gainesville, FL), ser-51 phosphorylated eIF2α (Abcam, Cambridge, UK), and eIF5. Secondary antibody probing and quantification was performed using IRDye 800CW goat anti-rabbit IgG with an Odyssey Fc imaging system (Li-Cor, Cambridge, United Kingdom).

Jennings M.D., Kershaw C.J., Adomavicius T, & Pavitt G.D. (2017). Fail-safe control of translation initiation by dissociation of eIF2α phosphorylated ternary complexes. eLife, 6, e24542.

+ Open protocol

+ Expand

Shipworm Rearing for Biological Studies

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Shipworms of the species Lyrodus pedicellatus from the Atlantic lineage [61 (link)] were used for our work. Samples were collected from the pier of Portsmouth Harbour (50° 47′ 47″ N, 1° 01′48″ W). Larvae from the original wood were used to infest logs of Scots pine, which were kept in tanks in the laboratories of the Institute of Marine Science, University of Portsmouth. To rear the animals, the water was taken directly from the Langstone Harbour (34 PSU salinity) and kept aerated and at a temperature of 15–18 °C using a flow-through system. The wood logs were opened by splitting them with a hammer and screwdriver, and the animals were then extracted with tweezers, placed in sea water containing EDTA-free protease inhibitors (1% v/v, Thermo Scientific) and kept on ice until dissection to anesthetise them. Species identification was performed using the pallets as described in [62 ]. The dissections were performed using a stereomicroscope (Leica MZ6) after removing the mantle to expose the organs.

Pesante G., Sabbadin F., Elias L., Steele-King C., Shipway J.R., Dowle A.A., Li Y., Busse-Wicher M., Dupree P., Besser K., Cragg S.M., Bruce N.C, & McQueen-Mason S.J. (2021). Characterisation of the enzyme transport path between shipworms and their bacterial symbionts. BMC Biology, 19, 233.

+ Open protocol

+ Expand

Purification of GST-tagged Human Rheb

Check if the same lab product or an alternative is used in the 5 most similar protocols

An N-terminal GST fusion protein of full length human Rheb (GST-Rheb) was expressed in Rosetta2 (DE3) E. coli (Novagen, Merck Millipore, Billerica, MA) using a pGEX-4T-2 vector (Tee et al., 2003 (link)), grown in 2xYT media and induced with 0.5 m IPTG at 37°C for 3 hr. Cells were resuspended in 20 mM Tris pH 8.0, 500 mM NaCl, 2 mM DTT with EDTA-free protease inhibitors (Pierce, Thermo Fisher Scientific) and lysed by sonication. Initial purification was achieved by glutathione Sepharose 4B (GE Healthcare, UK) affinity chromatography, with elution in 20 mM Tris pH 8.0, 200 mM NaCl, 2 mM DTT, 10 mM reduced glutathione. GST-Rheb was further purified by anion exchange chromatography (GE Healthcare), concentrated (Millipore Amicon Ultra-4), flash-frozen in liquid nitrogen and stored at −80°C in 20 mM Tris pH 8.0, 300 mM NaCl, 5 mM MgCl₂, 2 mM DTT at 4 mg/ml. Protein samples were analysed by SDS-PAGE using the Bolt Bis-Tris system with SimplyBlue SafeStain (Thermo Fisher). Protein concentrations were measured using a Nanodrop 1000 spectrophotometer (Thermo Scientific) with extinction coefficients and molecular weights determined by ExPASy ProtParam (Bairoch et al., 2005 (link))

Carroll B., Maetzel D., Maddocks O.D., Otten G., Ratcliff M., Smith G.R., Dunlop E.A., Passos J.F., Davies O.R., Jaenisch R., Tee A.R., Sarkar S, & Korolchuk V.I. (2016). Control of TSC2-Rheb signaling axis by arginine regulates mTORC1 activity. eLife, 5, e11058.

+ Open protocol

+ Expand

Purification and In Vitro Assay of INPP5K

Check if the same lab product or an alternative is used in the 5 most similar protocols

Wild-type and mutant GST-tagged full-length INPP5K was expressed in BL21 pLysS cells and purified on GSA beads (Thermo Fisher Scientific) in assay buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 10 mM MgCl₂) plus 1% Triton X-100 and EDTA-free protease inhibitors (Roche Diagnostics). After washing, aliquots of beads were run on Coomassie gels to determine the abundance of full-length fusion proteins. Beads bearing equal amounts of fusion proteins were incubated in assay buffer containing 135 μM PtdIns(4,5)P₂diC8, and free phosphate was measured using the Malachite Green assay kit (Echelon Biosciences). Results of three independent experiments were presented as mean ± standard deviation. To minimize variability between purifications, all constructs were freshly prepared and purified in parallel for each experiment, and beads used in the assay were afterward run on Coomassie gels to confirm equal protein loading.

Wiessner M., Roos A., Munn C.J., Viswanathan R., Whyte T., Cox D., Schoser B., Sewry C., Roper H., Phadke R., Marini Bettolo C., Barresi R., Charlton R., Bönnemann C.G., Abath Neto O., Reed U.C., Zanoteli E., Araújo Martins Moreno C., Ertl-Wagner B., Stucka R., De Goede C., Borges da Silva T., Hathazi D., Dell’Aica M., Zahedi R.P., Thiele S., Müller J., Kingston H., Müller S., Curtis E., Walter M.C., Strom T.M., Straub V., Bushby K., Muntoni F., Swan L.E., Lochmüller H, & Senderek J. (2017). Mutations in INPP5K, Encoding a Phosphoinositide 5-Phosphatase, Cause Congenital Muscular Dystrophy with Cataracts and Mild Cognitive Impairment. American Journal of Human Genetics, 100(3), 523-536.

+ Open protocol

+ Expand

Aspartic Acid Uptake and Protein Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells were seeded overnight (57,000 per well) in 6-well-plates with triplicate wells per condition.
The cells were then washed twice with PBS and incubated with either H-H or L-L medium supplemented with 1µCi per well of L-[U-14 C]-Aspartic acid (PerkinElmer, NEC268E050UC) for 40 h. The cells were washed twice with PBS before DNA was extracted using PureLink® Genomic DNA Mini Kit (Thermo Fisher-K182001) and quantified using a spectrophotometer. Equal volumes of DNA were added to scintillation vials and radioactivity was measured by liquid scintillation counting and normalized to total DNA concentration.
Immunoblotting. For western blotting, cells were rinsed once in ice-cold PBS and collected in lysis buffer containing 50 mM HEPES KOH, pH 7.4, 40mM NaCl, 2mM EDTA, 1.5mM orthovanadate, 50mM NaF, 10mM pyrophosphate, 10mM glycerophosphate, EDTA-free protease inhibitors (Thermo Fisher Scientific, 88266) and 1% Triton X-100. Proteins from total lysates were resolved by 8-12% SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) and the blot was exposed to film.

Tsai P., Lee M., Jadhav U., Naqvi I., Madha S., Adler A., Mistry M., Naumenko S., Lewis C.A., Hitchcock D.S., Roberts F.R., DelNero P., Hank T., Honselmann K.C., Oyarvide V.M., Mino‐Kenudson M., Clish C.B., Shivdasani R.A., & Kalaany N.Y. (2020). Adaptation of pancreatic cancer cells to nutrient deprivation is reversible and requires glutamine synthetase stabilization by mTORC1.

+ Open protocol

+ Expand

Affinity Purification of eIF3 Complex

Check if the same lab product or an alternative is used in the 5 most similar protocols

HEK293T cells were transfected for 48 h with plasmids encoding V5-tagged subunits of the eIF3 complex with the use of the X-tremeGENE 9 DNA Transfection Reagent. Cells were lysed in ice-cold lysis buffer [50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 5 mM MgCl₂, 1 mM dithiothreitol (DTT), 1% Triton X-100, and EDTA-free protease inhibitor (Roche)], and the RNA concentration of the lysates was determined with the use of a Qubit RNA BR assay kit (Thermo Fisher Scientific). Dynabeads Protein G (Thermo Fisher Scientific) were conjugated with antibodies to V5 by incubation overnight at 4°C in lysis buffer supplemented with 0.5% bovine serum albumin, and they were washed with lysis buffer three times before use. The cell lysates were incubated with the antibody-coated beads for 90 min at 4°C, and the resulting immunoprecipitates were washed three times with wash buffer [50 mM Tris–HCl (pH 7.5), 300 mM NaCl, 1 mM DTT, 0.1% Triton X-100]. RNA was purified from the immunoprecipitates with the use of a Direct-zol RNA Kit (Zymo Research) and the addition of ISOGEN (Nippon Gene) directly to the beads. The amount of purified RNA was measured with the use of a Qubit RNA HS assay kit (Thermo Fisher Scientific).

Ichihara K., Matsumoto A., Nishida H., Kito Y., Shimizu H., Shichino Y., Iwasaki S., Imami K., Ishihama Y, & Nakayama K.I. (2021). Combinatorial analysis of translation dynamics reveals eIF2 dependence of translation initiation at near-cognate codons. Nucleic Acids Research, 49(13), 7298-7317.

+ Open protocol

+ Expand

Synaptosomal Enrichment from Rat Cortex

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Synaptosome enriched preparations were isolated from 50 mg of brain cortex from four rats using the Syn-PER reagent protocol (Thermofisher, Waltham, MA, USA). Briefly, 50 mg of cortex from each rat was homogenized in Syn-PER reagent to which was added an EDTA free protease inhibitor (Thermofisher, Cat#. A32955). Synaptosomes were enriched in the P2 fraction after the last centrifugation, with P1 and S1 fractions enriched in myelin, nuclei, and any non-homogenate tissue or cytosolic elements. Resulting P2 proteins were re-suspended in Syn-PER and quantified via fluorometry using a DeNovix QFX unit (DeNovix, New Castle, DE, USA) and flowcytometry using a Guava EasyCyte (Guava Soft v2.7, Luminex, Austin, TX, USA) [35 (link)]. For the preparation of cerebellar synaptosome enriched preparations, 50 mg of the whole cerebellum was used following the same procedure described above.

Miller B., Moreno N., Gutierrez B.A, & Limon A. (2022). Microtransplantation of Postmortem Native Synaptic mGluRs Receptors into Xenopus Oocytes for Their Functional Analysis. Membranes, 12(10), 931.

+ Open protocol

+ Expand

Quantifying PIF7 Protein Levels in Seedlings

Check if the same lab product or an alternative is used in the 5 most similar protocols

For PIF7 protein analyses, two-wk-old seedlings grown in either LD or LD+FR were harvested at the indicated time points. Total proteins were extracted from frozen ground tissues in the extraction buffer [50 mM sodium phosphate (pH 7.4), 150 mM KCl, 10% glycerol, 5 mM EGTA, 0.1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 50 μM MG-132, 2 mM Na 3 VO 4 , 2 mM NaF, EDTA-free protease inhibitor (Thermo Fisher Scientific, Waltham, MA, USA), 1 mM DTT, and phosphatase inhibitor (Roche, Basel, Switzerland)]. Nuclear proteins were prepared using CelLytic Plant Nuclei Isolation/Extraction Kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer's protocol. HA-PIF7 proteins were detected using a monoclonal anti-HA (3F10, Roche) antibody. Actin or histone H3 proteins were detected by anti-actin (10-B3, Sigma-Aldrich) or antihistone H3 (MABI0301, MBL, Nagoya, Aichi, Japan) antibodies, respectively, followed by an HRP-conjugated goat anti-mouse antibody (MBL330, MBL). For protein quantification, Image Studio Lite (LI-COR, Lincoln, LE, USA) was used. Relative protein abundance was normalized against actin or histone H3. Relative PIF7 protein levels were further normalized against a geometric mean of the LD condition, and data from three biological replicates were presented.

Lee N., Ozaki Y., Hempton A.K., Takagi H., Purusuwashi S., Song Y.H., Endo M., Kubota A, & Imaizumi T. (2023). The FLOWERING LOCUS T gene expression is controlled by high-irradiance response and external coincidence mechanism in long days in Arabidopsis. The New phytologist, 239(1).

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!