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Complete mini edta

Manufactured by Roche
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Sourced in Germany

The Complete Mini-EDTA is a laboratory equipment product designed for the collection, storage, and transportation of small-volume blood samples. It contains EDTA as an anticoagulant to prevent blood clotting. The product is intended for professional use in clinical and research settings.

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Lab products found in correlation

Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Sep pak c18 cartridge, by Waters Corporation (1 mentions) Dithiothreitol (dtt), by Merck Group (1 mentions) Iodoacetamide, by Merck Group (1 mentions) Trypsin, by Promega (1 mentions) Phosphostop, by Roche (1 mentions) Nitrocellulose membrane, by GE Healthcare (1 mentions) Ab133327, by Abcam (1 mentions) Ab176840, by Abcam (1 mentions) Sc 40, by Santa Cruz Biotechnology (1 mentions) A4416, by Merck Group (1 mentions) A6154, by Merck Group (1 mentions) Bioruptor pico, by Diagenode (1 mentions) Poly di dc, by Merck Group (1 mentions) Pvdf western blotting membrane, by Roche (1 mentions) Sds page gel, by Thermo Fisher Scientific (1 mentions) Ripa buffer, by Merck Group (1 mentions) Chemidoc imaging system, by Bio-Rad (1 mentions) Clarity western ecl substrate, by Bio-Rad (1 mentions) Rc dc protein assay kit, by Bio-Rad (1 mentions)

13 protocols using complete mini edta

1

Breast Cancer Cell Lysis and Preparation for Proteomic Analysis

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Breast cancer cell lines were grown to 90 % confluency under indicated culture conditions (Supplementary Table 1). For cell lysis of adherent cells, growth media was removed and cells were rinsed with PBS before being trypsinized to remove from growth plastic. Cells were then counted and 3.0 x 106 cells were transferred to a new tube and pelleted. Media was aspirated and cells were washed with PBS. After re-pelleting cells, the PBS was aspirated and the cells were fast-frozen on dry ice then stored at -80 °C until lysis. For cells growing in suspension, cells were pelleted and re-suspended before counting. Again, 3.0 x 106 cells were pelleted, rinsed and frozen as before.
Cells were lysed with 200 μL of lysis buffer by passing through a 21 gauge needle 20 times. Lysis buffer was composed of 75 mM NaCl, 3 % SDS, 1 mM NaF, 1 mM beta-glycerophosphate, 1 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 1 mM PMSF and 1X Roche Complete Mini EDTA free protease inhibitors in 50 mM HEPES, pH 8.536 (link). Lysates were then sonicated for 5 minutes in a sonicating water bath before cellular debris was pelleted by centrifugation at 14000 rpm for 5 minutes.
Lapek JD J.r., Greninger P., Morris R., Amzallag A., Pruteanu-Malinici I., Benes C.H, & Haas W. (2017). Detection of Dysregulated Protein Association Networks by High-Throughput Proteomics Predicts Cancer Vulnerabilities. Nature biotechnology, 35(10), 983-989.
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2

Breast Cancer Cell Lysis and Preparation for Proteomic Analysis

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Breast cancer cell lines were grown to 90 % confluency under indicated culture conditions (Supplementary Table 1). For cell lysis of adherent cells, growth media was removed and cells were rinsed with PBS before being trypsinized to remove from growth plastic. Cells were then counted and 3.0 x 106 cells were transferred to a new tube and pelleted. Media was aspirated and cells were washed with PBS. After re-pelleting cells, the PBS was aspirated and the cells were fast-frozen on dry ice then stored at -80 °C until lysis. For cells growing in suspension, cells were pelleted and re-suspended before counting. Again, 3.0 x 106 cells were pelleted, rinsed and frozen as before.
Cells were lysed with 200 μL of lysis buffer by passing through a 21 gauge needle 20 times. Lysis buffer was composed of 75 mM NaCl, 3 % SDS, 1 mM NaF, 1 mM beta-glycerophosphate, 1 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 1 mM PMSF and 1X Roche Complete Mini EDTA free protease inhibitors in 50 mM HEPES, pH 8.536 (link). Lysates were then sonicated for 5 minutes in a sonicating water bath before cellular debris was pelleted by centrifugation at 14000 rpm for 5 minutes.
Lapek JD J.r., Greninger P., Morris R., Amzallag A., Pruteanu-Malinici I., Benes C.H, & Haas W. (2017). Detection of Dysregulated Protein Association Networks by High-Throughput Proteomics Predicts Cancer Vulnerabilities. Nature biotechnology, 35(10), 983-989.
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3

Phosphoproteomics of Th17 Cell Signaling

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Eight-day OT-II/Th17 cultures were subjected to a Ficoll gradient to remove cell debris and washed twice in IMDM-10% to remove remaining cytokines. Cells were cultured in IMDM-10%-2ME without polarizing cytokines for 4 h prior to stimulation with IL-23 at 10 ng/mL for 30 min. Phosphoproteomics was performed using approximately 60 × 106 cells in the three biological replicates (30 × 106 per condition). After stimulation, cells were pelleted, washed twice in ice-cold HBSS, and lysed at 50 × 106 cells/mL in 8 M urea, 50 mM Tris-HCl (pH 8), supplemented with protease and phosphatase inhibitor tablets (Complete Mini-EDTA and PhosphoStop; Roche). Lysates were vigorously shaken for 15 min/RT, sonicated, and shaken for additional 15 min/RT. The amount of extracted protein was determined by BCA assay (Pierce). Approximately 1 mg of protein was obtained per condition in each biological replicate. The protein lysates were then reduced (10 mM DTT; Sigma-Aldrich) and alkylated (50 mM iodoacetamide; Sigma-Aldrich) prior to overnight digestion at 37°C with trypsin (1:80 trypsin:protein, w/w; Promega). Digested proteins were desalted using C18 Sep-Pak cartridges (Waters) and subjected to dimethyl labeling.
Álvarez-Salamero C., Castillo-González R., Pastor-Fernández G., Mariblanca I.R., Pino J., Cibrian D, & Navarro M.N. (2020). IL-23 signaling regulation of pro-inflammatory T-cell migration uncovered by phosphoproteomics. PLoS Biology, 18(3), e3000646.
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4

Western Blot Analysis Protocol

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Supernatant samples were harvested by centrifugation of cultures at 1000 rpm. Cells were lysed in western lysis buffer (10 mM β‐glycerophosphate, 20 mM 4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid (HEPES), 100 mM NaCl, and 0.5% (w/v) Triton X‐100 with 1 mM NaV, 50 mM NaF supplemented with a protease inhibitor cocktail tablet; Roche cOmplete mini EDTA‐free 11836170001), denatured by addition of Laemmli's buffer and then electrophoresis undertaken after using a Bradford assay to determine equivalent protein loading (for cell lysates). Following transfer to nitrocellulose membranes (GE Healthcare), blots were probed with primary antibodies (see below), horseradish peroxidase‐conjugated secondary antibodies and enhanced chemiluminescence (ECL) (GE Healthcare) was used for detection essentially as previously described in (Roobol et al., 2011 (link)). ImageJ (NIH) was used for band quantification. Alternatively, gels were stained using Coomassie blue. Densitometry analysis was undertaken using ImageJ software.
Knight T.J., Povey J.F., Vito D., Mohindra A., Jaques C.M, & Smales C.M. (2022). Manipulation of mRNA translation elongation influences the fragmentation of a biotherapeutic Fc‐fusion protein produced in CHO cells. Biotechnology and Bioengineering, 119(12), 3408-3420.
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5

SDS-PAGE and Immunoblotting Protocol

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SDS–PAGE and immunoblotting was performed as described previously5 (link). In brief, cell pellets were lysed in RIPA buffer containing cOmplete Mini EDTA free (Roche) on ice for 20 min. Samples were then sonicated in a water-cooled Bioruptor Pico (Diagenode) and centrifuged at 21,000g for 15 min at 4 °C. Protein concentration was determined using a Pierce BCA protein assay kit (Thermo Fisher Scientific). Forty micrograms of protein was loaded per well. The primary antibodies were anti-CDK1 (1:1,000, ab133327, Abcam), anti-Myc (1:1,000, sc-40, Santa Cruz) and anti-histone H3.3 (1:5,000, ab176840, Abcam). The secondary antibodies were anti-mouse IgG peroxidase conjugate (1:10,000, A4416, Sigma-Aldrich) and anti-rabbit IgG peroxidase conjugate (1:10,000, A6154, Sigma-Aldrich).
Meijering A.E., Sarlós K., Nielsen C.F., Witt H., Harju J., Kerklingh E., Haasnoot G.H., Bizard A.H., Heller I., Broedersz C.P., Liu Y., Peterman E.J., Hickson I.D, & Wuite G.J. (2022). Nonlinear mechanics of human mitotic chromosomes. Nature, 605(7910), 545-550.
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6

NF-κB Activation Detected by EMSA

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Five million cells were lysed for 30 min in ice-cold electrophoretic mobility shift assay (EMSA) lysis buffer composed of 20 mM HEPES (pH 7.9), 350 mM NaCl, 1 mM MgCl2, 0.5 mM EDTA, 0.15 mM EGTA, 20% glycerol, 1% Nonidet P-40 (NP-40), 0.5 mM dithiothreitol (DTT), and protease inhibitor cocktail (1 tablet/10 mL of EMSA lysis buffer, cOmplete mini EDTA free; catalog number 11836170001; Roche) prior centrifugation at 14,000 × g for 10 min. Then, 1 mg of poly(dI-dC) (catalog number P4929; Sigma-Aldrich) and EMSA binding buffer (75 mM NaCl, 15 mM Tris HCl [pH 7.5], 1.5 mM EDTA, 1.5 mM DTT, 7.5% glycerol, 0.3% NP-40, 20 mg/mL BSA) were mixed with 10 mg of cell lysate in a total reaction volume of 9 μL for 20 min at 4°C. Next, 1 μl of [32P]-labeled double-stranded oligonucleotides (5′-TCAACAGAGGGGACTTTCCGAGAGGCC-3′) containing the underlined intronic κB site corresponding to the Igκ gene was added to the reaction mixture and incubated for 20 min at room temperature. Then, 4% native polyacrylamide gels were employed to separate the samples, which were later dried and exposed to film on a phosphor screen. NF-κB/κB complexes were visualized using Image Quant software.
Romero N., Tishchenko A., Verhamme R., Wuerzberger-Davis S.M., Van Waesberghe C., Nauwynck H.J., Miyamoto S, & Favoreel H.W. (2023). Several Alphaherpesviruses Interact Similarly with the NF-κB Pathway and Suppress NF-κB-Dependent Gene Expression. Microbiology Spectrum, 11(4), e01421-23.
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7

Western Blot Analysis of Protein Samples

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Cells were collected and lysed with RIPA buffer (Sigma) containing phosphatase inhibitor phosSTOP and protease inhibitors cOmplete mini EDTA-free (Roche). Protein concentration was quantified using RC DC™ Protein Assay Kit (Bio-Rad). Proteins were separated using SDS-PAGE Gels (Thermo Fisher) and transferred to PVDF Western Blotting membranes (Roche). The membranes were blocked with 5% bovine serum albumin (BSA, Sigma). Blots were incubated overnight with primary antibodies listed in the KEY RESOURCES table, followed by 1 h incubation with appropriate secondary antibody. Membranes were developed using SuperSignalTM West Pico Plus ECL substrate (Thermo Scientific) or ClarityTM Western ECL Substrate (Bio-Rad). Images were acquired using the Omega Lum™ C Imaging System (Gel Company) or ChemiDoc™ Imaging System (Bio-Rad).
Miglioranza Scavuzzi B., van Drongelen V., Kaur B., Fox J.C., Liu J., Mesquita-Ferrari R.A., Kahlenberg J.M., Farkash E.A., Benavides F., Miller F.W., Sawalha A.H, & Holoshitz J. (2022). The lupus susceptibility allele DRB1*03:01 encodes a disease-driving epitope. Communications Biology, 5, 751.
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8

GFP and P-eIF2α Immunoblotting Protocol

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For GFP blots, 5 L2 larvae were homogenized in 1X Laemmli/RIPA buffer containing 1X protease inhibitors. For P-eif2α blots, protein from S2 cells or brain/eye-imaginal discs was isolated in 1X Laemmli/RIPA buffer containing 1X protease inhibitors (Roche cOmplete Mini EDTA-free protease inhibitor tablets), as well as the phosphatase inhibitors Calyculin A and okadaic acid.
Equivalent amounts of protein were resolved by SDS-PAGE (10% acrylamide) and transferred to PVDF membrane by semi-dry transfer. Membranes were then treated with either 5% BSA or 5% milk protein block in 1XTBST prior to immunoblotting. Blots were probed with antibodies for GFP (1:5000, Thermo-Fisher #A6455), tubulin (1:2000, Developmental Studies Hybridoma Bank #12G10), P-eif2α (1:1000, abcam #32157), and Pan-eif2α (1:500, abcam #26197). Blots shown are representative of at least three biological replicates, and quantification was performed using Image J software.
Palu R.A, & Chow C.Y. (2018). Baldspot/ELOVL6 is a conserved modifier of disease and the ER stress response. PLoS Genetics, 14(8), e1007557.
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9

Western Blot Analysis of Pluripotency Factors

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Whole-cell extracts were prepared by sorted cells from day 4 embryoids (WT; TNAP/NANOS3-mCherry double positive population, BLIMP1 KO; TNAP positive population, SOX17 KO; whole population) in lysis buffer composed of 50mM Tris-HCl (pH7.5), 0.15M NaCl, 0.1% SDS, 1% Triton X-100, 1% Sodium deoxycholate and cOmplete mini EDTA free (Roche Applied Science, Penzberg, Germany). After electrophoresis, proteins were transferred to nitrocellulose membranes. Membranes were incubated in Western Blocking Reagent (Roche Applied Science) and treated with antibodies. Primary antibodies against BLIMP1 (rat IgG; eBioscience), SOX17 (goat IgG; R&D systems), and TUBULIN (mouse IgG; Sigma) were used. Horseradish peroxidase-conjugated secondary antibodies against rat, goat or rat IgG were added (Dako, Life technologies). After antibody treatment, blots were developed using ECL Western Blotting Detection System (GE Healthcare).
Irie N., Weinberger L., Tang W.W., Kobayashi T., Viukov S., Manor Y.S., Dietmann S., Hanna J.H, & Surani M.A. (2015). SOX17 Is a Critical Specifier of Human Primordial Germ Cell Fate. Cell, 160(1-2), 253-268.
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10

Protein Extraction and Western Blot

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The cells were harvested and then resuspended in modified RIPA buffer (10 mm Tris/HCl, pH 7.4, 1 mm EDTA, 150 mm NaCl, 1% Nonidet P‐40, 1% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitor cocktail (Complete Mini EDTA free from Roche). Then, the cells were lysed in RIPA for 20 min on ice and cell debris was removed by centrifugation. The proteins were resuspended in Laemmli buffer and separated by 10% SDS/PAGE. The gels were blotted to nitrocellulose membranes and immunoassayed using the indicated antibodies. The proteins were visualized by chemiluminescence using the application of AI‐600 (GE Healthcare, Chicago, IL, USA).
Ryabchenko B., Soldatova I., Šroller V., Forstová J, & Huérfano S. (2021). Immune sensing of mouse polyomavirus DNA by p204 and cGAS DNA sensors. The Febs Journal, 288(20), 5964-5985.
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