Complete edta free proteinase inhibitor cocktail tablet

Manufactured by Roche

Sourced in Switzerland

The Complete EDTA-free Proteinase Inhibitor Cocktail Tablet is a lab equipment product that contains a mixture of proteinase inhibitors formulated without EDTA. The primary function of this product is to prevent the degradation of proteins during cell lysis or protein extraction procedures.

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Lab products found in correlation

Anti gapdh, by Cell Signaling Technology (2 mentions) G box, by Syngene (2 mentions) Bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Anti pan ago, by Merck Group (1 mentions) Anti ha 11, by BioLegend (1 mentions) Pvdf membrane, by Bio-Rad (1 mentions) Western lightning plus ecl, by PerkinElmer (1 mentions) Anti sema7a, by Santa Cruz Biotechnology (1 mentions) Anti grb2, by Cell Signaling Technology (1 mentions) Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions) Nitrocellulose membrane, by Bio-Rad (1 mentions) Miracloth, by Merck Group (1 mentions)

5 protocols using complete edta free proteinase inhibitor cocktail tablet

Preparation of HEK293T In Vitro Translation Extracts

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In vitro translation extracts were made from HEK293T cells using a previously described protocol (Rakotondrafara and Hentze, 2011 (link)). Cells were scraped and collected by centrifugation for 2 minutes at 376 x g at 4°C. Cells were washed once with cold PBS (137mM NaCl, 2.7mM KCl, 100mM Na₂HPO₄, 2mM KH₂PO₄) then homogenized with an equal volume of freshly made cold hypotonic lysis buffer (10mM HEPES-KOH pH 7.6, 10mM KOAc, 0.5mM Mg(OAc)₂, 5mM dithiothreitol (DTT), and 1 Complete EDTA-free Proteinase Inhibitor Cocktail tablet (Roche) per 10 mL of buffer). After hypotonic-induced swelling for 45 min on ice, cells were homogenized using a syringe attached to a 27G needle until 95% of cells burst as determined by trypan blue staining. Lysate was then centrifuged at 14,000 x g for 1 min at 4°C. The resulting supernatant was moved to a new tube, avoiding the top lipid layer. Lysate aliquots were quickly frozen with liquid nitrogen and stored at −80°C.

Mendez A.S., Ly M., González-Sánchez A.M., Hartenian E., Ingolia N.T., Cate J.H, & Glaunsinger B.A. (2021). The N-terminal domain of SARS-CoV-2 nsp1 plays key roles in suppression of cellular gene expression and preservation of viral gene expression. Cell Reports, 37(3), 109841.

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Nuclear-Cytoplasmic Fractionation of C. elegans

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Nuclear/cytoplasmic fractionation was performed as described previously^{84 (link)}. Young adult worms were washed 3–5 times in M9, and twice in hypotonic buffer (15 mM HEPES, 10 mM KCl, 5 mM MgCl₂, 0.1 mM EDTA, 350 mM sucrose). Lysis was on ice in complete hypotonic buffer plus 1 mM DTT and 1 complete EDTA-free proteinase inhibitor cocktail tablet (Roche Applied Science) per 12 ml, using a motorized pellet pestle (Sigma Z359971, Z359947) until most worm carcasses were homogenized. Worm debris was pelleted at 500×g (2 × 5 min), and 5% of the resultant supernatant was kept as the input fraction. Nuclei were pelleted at 4000×g (5 min), and the resulting supernatant centrifuged again at 17,000×g and kept as the cytoplasmic fraction. Nuclear pellets were washed twice in complete hypotonic buffer and dissolved in complete hypertonic buffer (15 mM HEPES, 400 mM KCl, 5 mM MgCl₂, 0.1 mM EDTA, 0.1% Tween 20, 10% glycerol, 1 mM DTT, complete EDTA-free proteinase inhibitor as above).

Flynn S.M., Chen C., Artan M., Barratt S., Crisp A., Nelson G.M., Peak-Chew S.Y., Begum F., Skehel M, & de Bono M. (2020). MALT-1 mediates IL-17 neural signaling to regulate C. elegans behavior, immunity and longevity. Nature Communications, 11, 2099.

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Western Blot Analysis of Ago Proteins

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3 x 10⁷ of cells were lysed in 100 μl of NET-2 buffer supplemented with complete EDTA-free proteinase inhibitor cocktail tablet (Roche), sonicated as described above, and the supernatants were mixed with 35 μl 4X SDS-PAGE loading buffer. Typically, 25 μl (corresponding to ~40 μg total protein) were separated on a 10% SDS-PAGE gel, and electrotransferred to a PVDF membrane (BioRad). After blocking with 10% milk in 1× TBST (20 mM Tris [pH 7.5], 150 mM NaCl, 0.1% Tween 20), the membrane was probed with the appropriate antibodies and detected with Western Lightning Plus-ECL (PerkinElmer) using a GBox (SYNGENE). Primary antibodies used were anti-pan Ago (Sigma Millipore), anti-HA.11 (BioLegend) and anti-GAPDH (Cell Signaling).

Sheu-Gruttadauria J., Pawlica P., Klum S.M., Wang S., Yario T.A., Schirle Oakdale N.T., Steitz J.A, & MacRae I.J. (2019). Structural Basis for Target-Directed MicroRNA Degradation. Molecular cell, 75(6), 1243-1255.e7.

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Quantitative Western Blot Analysis

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Cell pellets were lysed in RIPA buffer (50 mM Tris-Cl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, supplemented with complete EDTA-free proteinase inhibitor cocktail tablet (Roche)), and quantified by Bradford assays using the Coomassie Plus Assay Kit (Pierce). Typically, 10 μg total protein was separated on a 12% SDS-PAGE gel, then transferred to nitrocellulose membrane (BioRad). After blocking with 5% milk in 1x TBST, the membrane was probed with the appropriate antibody, detected with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific) according to the manufacturer’s protocol, imaged using a GBox (SYNGENE) and quantified by GeneTools v. 4.02 (SYNGENE). We obtained standard curves for SEMA7A and GRB2 by making serial dilutions of total cell lysate. Both curves show a strong linear relationship between signal and the amount of cell lysate (data not shown). The Western blots presented in the paper were performed within the linear range. Primary antibodies used were anti-SEMA7A (sc-376149, Santa Cruz Biotech), anti-GRB2 (#3972, Cell Signaling), anti-GAPDH (#2118, Cell Signaling) and anti-TUBULIN (#CP06, DM1A, Calbiochem). GAPDH and TUBULIN were provided as normalization controls. Data shown in the figures are representative of at least three independent experiments.

Guo Y.E., Riley K.J., Iwasaki A, & Steitz J.A. (2014). Alternative Capture of Noncoding RNAs or Protein-Coding Genes by Herpesviruses to Alter Host T-Cell Function. Molecular cell, 54(1), 67-79.

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Isolation of Asparagus Microsomes

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Microsomes were isolated from Asparagus spears following the procedure of Zeng et al. [40 (link)] with minor modifications. Briefly, plant tissues were homogenized with extraction buffer (1 mL/g tissue) containing 50 mM HEPES-KOH (pH 6.8), 0.4 M sucrose, 1 mM dithiothreitol (DTT), 5 mM MnCl₂, 5mM MgCl₂ and complete EDTA-free proteinase inhibitor cocktail tablet (Roche, Basel, Switzerland) in a kitchen magic bullet blender. The homogenate was filtered through two layers of miracloth (Merck Millipore, Billerica, MA, US) and the filtrate was centrifuged at 3000g for 20 min at 4°C. The supernatant was centrifuged at 100,000g for 30 min at 4°C. The 100,000g pellets were resuspended in homogenization buffer and stored at -80°C. The protein concentration was measured using the BCA protein assay kit (Thermo Scientific, Rockford, IL, US). The bovine serum albumin (BSA) provided in the kit was used as the standard.

Song L., Zeng W., Wu A., Picard K., Lampugnani E.R., Cheetamun R., Beahan C., Cassin A., Lonsdale A., Doblin M.S, & Bacic A. (2015). Asparagus Spears as a Model to Study Heteroxylan Biosynthesis during Secondary Wall Development. PLoS ONE, 10(4), e0123878.

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