edta free protease inhibitor cocktail, by Roche - Product details

1

Sialidase Activity and Protein Separation

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Example 11

Purified bacteria sialidases (1 μg) were incubated with DFSA (0.1 mM) at room temperature for 1 h, and separated on 4-12% NuPAGE (Invitrogen). 5×105 influenza viruses were incubated with DFSA (0.1 mM) at room temperature for 1 h, and separated on 4-12% NuPAGE (Invitrogen). Sialidase transfectant 293T cells were lysed by different lysis buffers: pH4.5 (1% NP-40, 100 mM NaOAc, 150 mM NaCl, 3 mM KCl, pH 4.5, 1×EDTA-free protease inhibitor cocktail from Roche), pH 7.4 buffer (1% NP-40, 25 mM Tris, 150 mM NaCl, 3 mM KCl, pH 7.4, 1×EDTA-free protease inhibitor cocktail from Roche), and pH 9.0 buffer (1% NP-40, 25 mM Tris, 150 mM NaCl, 3 mM KCl, pH 9.0, 1×EDTA-free protease inhibitor cocktail from Roche). The lysates were collected and incubated with DFSA (0.1 mM) at 37° C. for 1 h. Following incubation, the samples were clarified, and protein concentrations were determined by bicinchoninic acid protein assay kit (Pierce). For each sample, 20 μg total lysate was separated on 4-12% NuPAGE (Invitrogen). After electrophoresis, the gels were blotted onto PVDF membranes (Millipore). Click reactions were performed on the PVDF membranes, and labeling signals were processed and analyzed by chemiluminescence detector.

US10214765B2. Cell-permeable probes for identification and imaging of sialidases (2019-02-26). Academina Sinica [TW]. Inventors: Chi-Huey Wong [US], Jim-Min Fang [TW], Yih-Shyun E. Cheng [TW], Chamg-Sheng Tsai [TW].

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2

Ribosomal Subunit Dissociation and Re-association Assay

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This assay was adapted from a previously published work (Burwick et al., 2012 (link)). For ribosomal subunit dissociation, 20~30 TF-1 cells were harvested without cycloheximide treatment and lysed with low Mg²⁺ buffer (20mM Tris, pH7.4, 140mM KCl, 0.25mM MgCl₂, 0.5mM DTT, 1% Triton X-100, EDTA-free protease inhibitor cocktail (Roche)) for 10min on ice. After clarification by centrifugation at 17,000g for 5min at 4°C, the cell lysate was divided into two aliquots. One aliquot was loaded on a 7–45% low Mg²⁺ sucrose gradient (20mM Tris, pH7.4, 140mM KCl, 0.25mM MgCl₂, 0.5mM DTT, EDTA-free protease inhibitor cocktail (Roche)) and analyzed with a BioComp fractionator to detect total 40S and 60S.
For ribosomal subunit re-association, 2.5M MgCl₂ was added to the other aliquot for a final concentration of 10mM Mg²⁺ and incubated for 5min at 37°C. The resultant cell lysate was loaded on a 7–45% high Mg²⁺ sucrose gradient (20mM Tris, pH7.4, 140mM KCl, 10mM MgCl₂, 0.5mM DTT, EDTA-free protease inhibitor cocktail (Roche)) and analyzed with a BioComp fractionator.

Lv K., Gong C., Antony C., Han X., Ren J.G., Donaghy R., Cheng Y., Pellegrino S., Warren A.J., Paralkar V.R, & Tong W. (2021). HectD1 Controls Hematopoietic Stem Cell Regeneration by Coordinating Ribosome Assembly and Protein Synthesis. Cell stem cell, 28(7), 1275-1290.e9.

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3

DDB2 Modification Detection in UV-C Irradiated Cells

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IP experiments were performed under denaturing conditions to detect DDB2 modifications. VH10 GFP-DDB2 cells were grown to confluency on 10 cm dishes and lysed 15 min after UV-C irradiation (30 J/m²) in lysis buffer (20 mM Tris-HCl pH 7.5, 50 mM NaCl, 0.5% NP-40, 1% SDS, 5 mM MgCl₂ and EDTA-free protease inhibitor cocktail (Roche)). Cell lysates were incubated with benzonase buffer (20 mM Tris-HCl pH 7.5, 50 mM NaCl, 0.5% NP-40, 0.5% Sodium Deoxycholate, 0.5% SDS, EDTA-free protease inhibitor cocktail (Roche) and 0.25 U/μL benzonase (Millipore)) for 45 min at room temperature in a tube rotator for digestion of chromatin. The suspension was spun down (15.000 g for 10 min) and the supernatant (Input) was used for GFP-DDB2 IP (GFP-DDB2 IP), by incubation of GFP-trap beads (Chromotek) for 2 h at room temperature. Beads were washed 5× (20 mM Tris-HCl pH 7.5, 50 mM NaCl, 0.5% NP-40, 0.5% Sodium Deoxycholate, 0.5% SDS and EDTA-free protease inhibitor cocktail (Roche)) and elution of immunoprecipitated proteins was performed by boiling the GFP-trap beads in 2× sample buffer for 5 min at 98 °C. Input and GFP-DDB2 IP fractions were analyzed by immunoblotting.

Ribeiro-Silva C., Sabatella M., Helfricht A., Marteijn J.A., Theil A.F., Vermeulen W, & Lans H. (2020). Ubiquitin and TFIIH-stimulated DDB2 dissociation drives DNA damage handover in nucleotide excision repair. Nature Communications, 11, 4868.

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4

Tandem Affinity Purification of Protein Complexes

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SILAC labeling reagents (89982 and 89990) were purchased from Thermo Scientific and cells were labeled according to the manufacturer’s instructions. Tandem affinity purification (TAP) or anti-Flag immune-precipitation (IP) was described previously^{17 (link)}. Briefly, cells were lysed in lysis buffer (150 mM NaCl, 10% glycerol, 50 mM Tris pH 7.5, 0.5% NP40, 5 mM EDTA, Roche EDTA-free protease inhibitor cocktail) at 4°C for 30 minutes, followed by centrifugation at 13,000×g for 15 minutes at 4°C. The supernatants were incubated with anti-Flag beads (Sigma, A2220) at 4°C for 3 hours. Beads were washed three times with lysis buffer and then once with HA buffer (150 mM NaCl, 10% glycerol, 50 mM Tris pH 7.5, 0.05% NP40, 1 mM EDTA, Roche EDTA-free protease inhibitor cocktail) followed by elution with Flag peptide (0.5 mg ml⁻¹) in HA buffer. FLAG elution samples were incubated with anti-HA beads (Santa Cruz Biotechnology, sc-7392AC) at 4°C for 2 hours. Beads were washed three times with HA buffer followed by elution with HA peptide (0.4 mg ml⁻¹) in HA buffer.

Yoda A., Adelmant G., Tamburini J., Chapuy B., Shindoh N., Yoda Y., Weigert O., Kopp N., Wu S.C., Kim S.S., Liu H., Tivey T., Christie A.L., Elpek K.G., Card J., Gritsman K., Gotlib J., Deininger M.W., Makishima H., Turley S.J., Javidi-Sharifi N., Maciejewski J.P., Jaiswal S., Ebert B.L., Rodig S.J., Tyner J.W., Marto J.A., Weinstock D.M, & Lane A.A. (2014). Mutations in G protein beta subunits promote transformation and kinase inhibitor resistance. Nature medicine, 21(1), 71-75.

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5

Tandem Affinity Purification of Protein Complexes

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SILAC labeling reagents (89982 and 89990) were purchased from Thermo Scientific and cells were labeled according to the manufacturer’s instructions. Tandem affinity purification (TAP) or anti-Flag immune-precipitation (IP) was described previously^{17 (link)}. Briefly, cells were lysed in lysis buffer (150 mM NaCl, 10% glycerol, 50 mM Tris pH 7.5, 0.5% NP40, 5 mM EDTA, Roche EDTA-free protease inhibitor cocktail) at 4°C for 30 minutes, followed by centrifugation at 13,000×g for 15 minutes at 4°C. The supernatants were incubated with anti-Flag beads (Sigma, A2220) at 4°C for 3 hours. Beads were washed three times with lysis buffer and then once with HA buffer (150 mM NaCl, 10% glycerol, 50 mM Tris pH 7.5, 0.05% NP40, 1 mM EDTA, Roche EDTA-free protease inhibitor cocktail) followed by elution with Flag peptide (0.5 mg ml⁻¹) in HA buffer. FLAG elution samples were incubated with anti-HA beads (Santa Cruz Biotechnology, sc-7392AC) at 4°C for 2 hours. Beads were washed three times with HA buffer followed by elution with HA peptide (0.4 mg ml⁻¹) in HA buffer.

Yoda A., Adelmant G., Tamburini J., Chapuy B., Shindoh N., Yoda Y., Weigert O., Kopp N., Wu S.C., Kim S.S., Liu H., Tivey T., Christie A.L., Elpek K.G., Card J., Gritsman K., Gotlib J., Deininger M.W., Makishima H., Turley S.J., Javidi-Sharifi N., Maciejewski J.P., Jaiswal S., Ebert B.L., Rodig S.J., Tyner J.W., Marto J.A., Weinstock D.M, & Lane A.A. (2014). Mutations in G protein beta subunits promote transformation and kinase inhibitor resistance. Nature medicine, 21(1), 71-75.

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6

Virus Purification and Cell Lysis Protocol

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Cells were briefly rinsed with ice cold PBS and irradiated once for HEK 293T cells, or twice for MT4 cells, with 0.15 J/cm² UV (λ = 365 nm) in a Stratalinker 2400 UV crosslinker (Stratagene). Cell culture supernatants containing virions were harvested and filtered with a 0.22 μm Steriflip filter unit (Millipore) and virions were purified through a 20% sucrose cushion in PBS by ultracentrifugation at 96,467 ×g (avg) in a SW28 rotor for 2 h, 4°C. Virus pellets were resuspended in 500 μl PBS and irradiated twice with 0.15 J/cm² UV (λ = 365 nm). Cells and virions were lysed in NP-40 lysis buffer (50 mM HEPES [pH 7.5], 150 mM KCl, 2 mM EDTA, 0.5% [v/v] NP-40, 1 mM dithiothreitol [DTT], 1× EDTA-free protease inhibitor cocktail [Roche]). After 30 min lysis on ice, the soluble fraction was separated by centrifugation at 17,000 × g for 5 min, 4°C. For lysates that contained A3F-3×HA the soluble lysate was adjusted to 1% NP-40, 0.25% Na-deoxycholate (Sigma) and 1% SDS and incubated for 15 min at 4°C, then the lysate was diluted 1:5 with dilution buffer (50 mM HEPES [pH 7.5], 150 mM KCl, 2 mM EDTA, 1 mM dithiothreitol [DTT], 1× EDTA-free protease inhibitor cocktail [Roche]). Lysates were subsequently treated with 20 U/ml of RNase A (Fermentas) and 60 U/ml with DNaseI (Roche) for 5 min at 37°C and then transferred to ice.

York A., Kutluay S.B., Errando M, & Bieniasz P.D. (2016). The RNA Binding Specificity of Human APOBEC3 Proteins Resembles That of HIV-1 Nucleocapsid. PLoS Pathogens, 12(8), e1005833.

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7

Immunoprecipitation of ATAD3 Complexes

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Two mg of mitochondria isolated from HEK293T cells were extracted in 500 μL of extraction buffer (20 mM HEPES, pH 7.4, 150 mM KCl, 0.5 mM PMSF, EDTA-free protease inhibitor cocktail (Roche), 1% digitonin, and 10 mM MgCl₂). Mitochondrial proteins were incubated with α-IgG (control), or α-ATAD3 conjugated Dynabeads (Life Technologies) at room temperature for 4 h. Following several antibody tests, the rabbit polyclonal anti-ATAD3 antibody-N-terminal from Abcam yielded efficient immunoprecipitation results. The supernatant containing unbound material was subsequently removed and the beads were washed 5 times with low-salt NET-2 buffer (50mM Tris-HCl pH 7.4, 150 mM NaCl, 0.05% NP-40, EDTA-free protease inhibitor cocktail (Roche)), or high-salt NET-2 buffer (with 300 mM NaCl), and once with 1X PBS. The bound proteins were eluted using dye-less buffer, precipitated with methanol/chloroform, and analyzed by mass spectrometry at the Keck Biotechnology Resource Laboratory (Yale University School of Medicine, New Haven, CT) as described (Tu and Barrientos, 2015 (link)). To analyze the interactome, hits with higher difference between IgG control and found ATAD3A were used for further analysis. Go terms were identified with enrichment analysis using gene list Enrichr analysis tool.

Arguello T., Peralta S., Antonicka H., Gaidosh G., Diaz F., Tu Y.T., Garcia S., Shiekhattar R., Barrientos A, & Moraes C.T. (2021). ATAD3A has a scaffolding role regulating mitochondria inner membrane structure and protein assembly. Cell reports, 37(12), 110139.

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8

Immunoprecipitation of ATAD3 Complexes

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Two mg of mitochondria isolated from HEK293T cells were extracted in 500 μL of extraction buffer (20 mM HEPES, pH 7.4, 150 mM KCl, 0.5 mM PMSF, EDTA-free protease inhibitor cocktail (Roche), 1% digitonin, and 10 mM MgCl₂). Mitochondrial proteins were incubated with α-IgG (control), or α-ATAD3 conjugated Dynabeads (Life Technologies) at room temperature for 4 h. Following several antibody tests, the rabbit polyclonal anti-ATAD3 antibody-N-terminal from Abcam yielded efficient immunoprecipitation results. The supernatant containing unbound material was subsequently removed and the beads were washed 5 times with low-salt NET-2 buffer (50mM Tris-HCl pH 7.4, 150 mM NaCl, 0.05% NP-40, EDTA-free protease inhibitor cocktail (Roche)), or high-salt NET-2 buffer (with 300 mM NaCl), and once with 1X PBS. The bound proteins were eluted using dye-less buffer, precipitated with methanol/chloroform, and analyzed by mass spectrometry at the Keck Biotechnology Resource Laboratory (Yale University School of Medicine, New Haven, CT) as described (Tu and Barrientos, 2015 (link)). To analyze the interactome, hits with higher difference between IgG control and found ATAD3A were used for further analysis. Go terms were identified with enrichment analysis using gene list Enrichr analysis tool.

Arguello T., Peralta S., Antonicka H., Gaidosh G., Diaz F., Tu Y.T., Garcia S., Shiekhattar R., Barrientos A, & Moraes C.T. (2021). ATAD3A has a scaffolding role regulating mitochondria inner membrane structure and protein assembly. Cell reports, 37(12), 110139.

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9

Purification of XIAP-Binding Proteins

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S10 cytoplasmic lysate from HeLa cells was prepared as described.¹⁷ (link) Briefly, HeLa-S cells (2 ml packed cell volume (PCV); Biovest International, Tampa, FL, USA) were resuspended in 2 ml of hypotonic buffer [10 mM Tris-HCl (pH 7.6), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT] containing EDTA-free protease inhibitor cocktail (Roche) and lysed using dounce homogenizer (30 strokes, pestle B). A mixture of 4 ml of HeLa S10 cytoplasmic lysate and 12 ml binding buffer [20 mM Tris (pH 7.6), 10 mM MgCl₂, 120 mM KCl, 8% sucrose, 2 mM DTT] containing EDTA-free protease inhibitor cocktail (Roche) and ribonuclease inhibitor (Promega) was incubated at 37°C for 10 min. An in vitro transcribed, strepto-tagged XIAP IRES RNA or strepto-tagged GAPDH RNA was added to the mixture and further incubated for 10 min at 37°C. RNA-dihydrostreptomycin affinity chromatography was performed as described^15,18 (link) and the RNA associated proteins were analyzed using protein gel blot analysis.

Thakor N., Smith M.D., Roberts L., Faye M.D., Patel H., Wieden H.J., Cate J.H, & Holcik M. (2016). Cellular mRNA recruits the ribosome via eIF3-PABP bridge to initiate internal translation. RNA Biology, 14(5), 553-567.

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10

Affinity Purification of DDX28 Complexes

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A stable pool of HEK293T cells expressing HA-6xHis-tagged DDX28 was established by puromycin (2 μg/ml) selection for 3 weeks after transfection with the construct DDX28- HA-6xHis/pIRESpuro2. Two mg of mitochondria isolated from this cell line or from HEK293T cells were extracted in 500 μl of extraction buffer (20 mM HEPES, pH 7.4, 0.5 mM PMSF, EDTA-free protease inhibitor cocktail (Roche), 1% digitonin, and 10 mM MgCl₂) containing 12.5, 150 or 300 mM KCl. Mitochondrial proteins were incubated with α-IgG (control), or α-HA-conjugated Dynabeads (Life Technologies) at room temperature for 4h. The supernatant containing unbound material was subsequently collected and the beads were washed 3 times with low-salt NET-2 buffer (50 mM Tris–HCl pH 7.4, 12.5 mM NaCl, 0.05% NP-40, EDTA-free protease inhibitor cocktail (Roche)), twice with high-salt NET-2 buffer (with 150 mM NaCl) and once with 1× PBS. The bound proteins were eluted using 50 μl of dye-less Laemmli buffer, precipitated with methanol/chloroform and analyzed by mass spectrometry at the Keck Biotechnology Resource Laboratory (Yale University School of Medicine, New Haven, CT, USA).

Maiti P., Kim H.J., Tu Y.T, & Barrientos A. (2018). Human GTPBP10 is required for mitoribosome maturation. Nucleic Acids Research, 46(21), 11423-11437.

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Edta free protease inhibitor cocktail

Lab products found in correlation

385 protocols using edta free protease inhibitor cocktail

Sialidase Activity and Protein Separation

Ribosomal Subunit Dissociation and Re-association Assay

DDB2 Modification Detection in UV-C Irradiated Cells

Tandem Affinity Purification of Protein Complexes

Tandem Affinity Purification of Protein Complexes

Virus Purification and Cell Lysis Protocol

Immunoprecipitation of ATAD3 Complexes

Immunoprecipitation of ATAD3 Complexes

Purification of XIAP-Binding Proteins

Affinity Purification of DDX28 Complexes

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