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Roche 1x roche protease inhibitor cocktail
1x Roche Protease Inhibitor Cocktail, supplied by Roche, used in various techniques. Bioz Stars score: 93/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/1x roche protease inhibitor cocktail/product/Roche
Average 93 stars, based on 12 article reviews
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1x roche protease inhibitor cocktail - by Bioz Stars, 2020-04
93/100 stars

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Transduction:

Article Title: Modulation of ?-Secretase Activity by Multiple Enzyme-Substrate Interactions: Implications in Pathogenesis of Alzheimer's Disease
Article Snippet: Preparation of cell membranes with γ-secretase (i.e. microsomal fractions) MEF cells, or MEF double knockout for endogenous presenilin transduced with human WT, dE9 and G384A presenilin 1 , were grown to confluence, scraped, and collected in pellets by centrifugation at 1000×g for 5 min. .. The cell pellets were re-suspended in 20 mM Pipes pH = 7.0, 140 mM KCl, 0.25 M sucrose, 5 mM EGTA, plus 1X Roche protease inhibitors cocktail, so that the total protein concentration was 10 mg/ml.

Clone Assay:

Article Title: A synthetic biology approach identifies the mammalian UPR RNA ligase RtcB
Article Snippet: To reconstitute XBP1u splicing in vitro , we first cloned a cDNA fragment encodingV5-XBP1uin -Rn luciferase into a pBluescript vector downstream of T7 promoter. .. Cell pellets were lysed in a buffer containing 30 mMTris-HCl, pH 7.4, 150 mMNaCl, 2mM MgCl2 , 0.5% Triton X-100, 5 mM β-mercaptoethanol, 1 mM PMSF, 1X Roche protease inhibitor cocktail and 1 X Sigma phosphatase inhibitor cocktail.

Centrifugation:

Article Title: Calcineurin Targets Involved in Stress Survival and Fungal Virulence
Article Snippet: The cell lysates were then further cleared of membrane-associated material by a second round of centrifugation at 14,000 rpm for 20 minutes. .. The beads were resuspended in ice-cold PBS with 1X Roche protease inhibitor cocktail, and 1 mM PMSF added and submitted to the Duke Proteomic Core Facility for phosphopeptide enrichment and phosphosite determination.

Article Title: Modulation of ?-Secretase Activity by Multiple Enzyme-Substrate Interactions: Implications in Pathogenesis of Alzheimer's Disease
Article Snippet: Preparation of cell membranes with γ-secretase (i.e. microsomal fractions) MEF cells, or MEF double knockout for endogenous presenilin transduced with human WT, dE9 and G384A presenilin 1 , were grown to confluence, scraped, and collected in pellets by centrifugation at 1000×g for 5 min. .. The cell pellets were re-suspended in 20 mM Pipes pH = 7.0, 140 mM KCl, 0.25 M sucrose, 5 mM EGTA, plus 1X Roche protease inhibitors cocktail, so that the total protein concentration was 10 mg/ml.

Luciferase:

Article Title: A synthetic biology approach identifies the mammalian UPR RNA ligase RtcB
Article Snippet: To reconstitute XBP1u splicing in vitro , we first cloned a cDNA fragment encodingV5-XBP1uin -Rn luciferase into a pBluescript vector downstream of T7 promoter. .. Cell pellets were lysed in a buffer containing 30 mMTris-HCl, pH 7.4, 150 mMNaCl, 2mM MgCl2 , 0.5% Triton X-100, 5 mM β-mercaptoethanol, 1 mM PMSF, 1X Roche protease inhibitor cocktail and 1 X Sigma phosphatase inhibitor cocktail.

Blocking Assay:

Article Title: The chemotherapeutic drug oxaliplatin differentially affects blood DC function dependent on environmental cues
Article Snippet: Preparation of protein lysates and Western blotting 2 × 105 cells were lysed in 20 μl lysis buffer containing 10 mM Tris/HCl pH 7.8, 5 mM EDTA, 50 mM NaCl, 1 mM Na3 VO4 10 mM pyrophosphate, 50 mM NaF, 1% Triton X-100, 1 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin and 1X Roche protease inhibitor cocktail (Roche Diagnostics Nederland BV, Almere, the Netherlands). .. After blocking, membranes were incubated with one the following antibodies: mouse-monoclonal-anti-β-actin (1:20,000; Sigma-Aldrich, St. Louis, MO), purified mouse-anti-PTP1C/Shp-1 (1:250; BD), rabbit-polyclonal-anti-STAT1, rabbit-polyclonal-anti-pSTAT1, rabbit-monoclonal-anti-STAT3 and rabbit-monoclonal-anti-pSTAT3 (all from Cell Signaling).

Article Title: Platinum-based drugs disrupt STAT6-mediated suppression of immune responses against cancer in humans and mice
Article Snippet: Regardless of cell type, 1 × 106 cells were lysed in 100 μl phosphatase-inhibiting lysis buffer containing 10 mM Tris/HCl, pH 7.8, 5 mM EDTA, 50 mM NaCl, 1 mM Na3 VO4 , 10 mM pyrophosphate, 50 mM NaF, 1% Triton X-100, 1 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 1X Roche protease inhibitor cocktail (Roche Diagnostics Nederland BV). .. After blocking, membranes were incubated with one of the following antibodies: mouse monoclonal anti–β-actin (1:10,000; Sigma-Aldrich), mouse monoclonal anti-pSTAT6 (pY641, 1:250; BD Biosciences — Pharmingen), or rabbit polyclonal anti-STAT6 antibody (S-20, 1:500; Santa Cruz Biotechnology Inc.).

Incubation:

Article Title: Calcineurin Targets Involved in Stress Survival and Fungal Virulence
Article Snippet: 30 μL aliquots of the equilibrated beads were added to the cell lysates, and the lysate + bead suspensions were incubated with constant rotation at 4°C for 2 hours. .. The beads were resuspended in ice-cold PBS with 1X Roche protease inhibitor cocktail, and 1 mM PMSF added and submitted to the Duke Proteomic Core Facility for phosphopeptide enrichment and phosphosite determination.

Article Title: The chemotherapeutic drug oxaliplatin differentially affects blood DC function dependent on environmental cues
Article Snippet: Preparation of protein lysates and Western blotting 2 × 105 cells were lysed in 20 μl lysis buffer containing 10 mM Tris/HCl pH 7.8, 5 mM EDTA, 50 mM NaCl, 1 mM Na3 VO4 10 mM pyrophosphate, 50 mM NaF, 1% Triton X-100, 1 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin and 1X Roche protease inhibitor cocktail (Roche Diagnostics Nederland BV, Almere, the Netherlands). .. After blocking, membranes were incubated with one the following antibodies: mouse-monoclonal-anti-β-actin (1:20,000; Sigma-Aldrich, St. Louis, MO), purified mouse-anti-PTP1C/Shp-1 (1:250; BD), rabbit-polyclonal-anti-STAT1, rabbit-polyclonal-anti-pSTAT1, rabbit-monoclonal-anti-STAT3 and rabbit-monoclonal-anti-pSTAT3 (all from Cell Signaling).

Article Title: Platinum-based drugs disrupt STAT6-mediated suppression of immune responses against cancer in humans and mice
Article Snippet: Regardless of cell type, 1 × 106 cells were lysed in 100 μl phosphatase-inhibiting lysis buffer containing 10 mM Tris/HCl, pH 7.8, 5 mM EDTA, 50 mM NaCl, 1 mM Na3 VO4 , 10 mM pyrophosphate, 50 mM NaF, 1% Triton X-100, 1 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 1X Roche protease inhibitor cocktail (Roche Diagnostics Nederland BV). .. After blocking, membranes were incubated with one of the following antibodies: mouse monoclonal anti–β-actin (1:10,000; Sigma-Aldrich), mouse monoclonal anti-pSTAT6 (pY641, 1:250; BD Biosciences — Pharmingen), or rabbit polyclonal anti-STAT6 antibody (S-20, 1:500; Santa Cruz Biotechnology Inc.).

Double Knockout:

Article Title: Modulation of ?-Secretase Activity by Multiple Enzyme-Substrate Interactions: Implications in Pathogenesis of Alzheimer's Disease
Article Snippet: Preparation of cell membranes with γ-secretase (i.e. microsomal fractions) MEF cells, or MEF double knockout for endogenous presenilin transduced with human WT, dE9 and G384A presenilin 1 , were grown to confluence, scraped, and collected in pellets by centrifugation at 1000×g for 5 min. .. The cell pellets were re-suspended in 20 mM Pipes pH = 7.0, 140 mM KCl, 0.25 M sucrose, 5 mM EGTA, plus 1X Roche protease inhibitors cocktail, so that the total protein concentration was 10 mg/ml.

Western Blot:

Article Title: The chemotherapeutic drug oxaliplatin differentially affects blood DC function dependent on environmental cues
Article Snippet: .. Preparation of protein lysates and Western blotting 2 × 105 cells were lysed in 20 μl lysis buffer containing 10 mM Tris/HCl pH 7.8, 5 mM EDTA, 50 mM NaCl, 1 mM Na3 VO4 10 mM pyrophosphate, 50 mM NaF, 1% Triton X-100, 1 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin and 1X Roche protease inhibitor cocktail (Roche Diagnostics Nederland BV, Almere, the Netherlands). ..

Article Title: Dephosphorylation is the Mechanism of Fibroblast Growth Factor Inhibition of Guanylyl Cyclase-B
Article Snippet: Briefly, ~200–500 μg crude membrane protein was diluted to 0.5 or 1 ml in 50 mM Tris-HCl pH 7.5, 50 mM NaF, 10 mM NaH2 PO4 , 2 mM EDTA, 0.5% deoxycholate, 0.1% SDS, 1% NP-40, 100 mM NaCl, 10 mM NaH2 PO4 , 1X Roche Protease Inhibitor Cocktail, and 1 μM microcystin. .. Phos-tag gel electrophoresis and western blotting were then performed as described, using a primary antibody made against the extracellular domain of GC-B [ ].

Article Title: Platinum-based drugs disrupt STAT6-mediated suppression of immune responses against cancer in humans and mice
Article Snippet: Paragraph title: Preparation of protein lysates and Western blotting. ... Regardless of cell type, 1 × 106 cells were lysed in 100 μl phosphatase-inhibiting lysis buffer containing 10 mM Tris/HCl, pH 7.8, 5 mM EDTA, 50 mM NaCl, 1 mM Na3 VO4 , 10 mM pyrophosphate, 50 mM NaF, 1% Triton X-100, 1 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 1X Roche protease inhibitor cocktail (Roche Diagnostics Nederland BV).

Protease Inhibitor:

Article Title: Histone deacetylases 1 and 2 maintain S-phase chromatin and DNA replication fork progression
Article Snippet: .. Nuclei were stored in nuclei storage buffer (50 mM Tris- HCl, pH 8.3, 40% glycerol, 0.1 mM EDTA, 5 mM Mg(CH3COO)2, 5 mM DTT and 1X Roche protease inhibitor cocktail) and stored at −80°C until use. .. Immunoprecipitation Nuclear extract was prepared in RIPA buffer supplemented with protease inhibitors (Roche) and precleared with Protein A-agarose beads (Millipore, Billerica, MA, USA) for 20 min at 4°C with constant rotation.

Article Title: Calcineurin Targets Involved in Stress Survival and Fungal Virulence
Article Snippet: .. The beads were resuspended in ice-cold PBS with 1X Roche protease inhibitor cocktail, and 1 mM PMSF added and submitted to the Duke Proteomic Core Facility for phosphopeptide enrichment and phosphosite determination. ..

Article Title: The chemotherapeutic drug oxaliplatin differentially affects blood DC function dependent on environmental cues
Article Snippet: .. Preparation of protein lysates and Western blotting 2 × 105 cells were lysed in 20 μl lysis buffer containing 10 mM Tris/HCl pH 7.8, 5 mM EDTA, 50 mM NaCl, 1 mM Na3 VO4 10 mM pyrophosphate, 50 mM NaF, 1% Triton X-100, 1 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin and 1X Roche protease inhibitor cocktail (Roche Diagnostics Nederland BV, Almere, the Netherlands). ..

Article Title: Dephosphorylation is the Mechanism of Fibroblast Growth Factor Inhibition of Guanylyl Cyclase-B
Article Snippet: .. RCS cells were lysed for 30 min at 4°C on a rotator in RIPA buffer containing: 50 mM HEPES pH 7.4, 50 mM NaF, 2 mM EDTA, 0.5% deoxycholate, 0.1% SDS, 1% IGEPAL CA-630, 100 mM NaCl, 10 mM NaH2 PO4 , 1X Roche Protease Inhibitor Cocktail, and 0.5 μM microcystin. .. Cellular extracts were then precleared on a rotator in the same RIPA buffer at 4 °C containing 50 μl IPA300 Protein A-conjugated resin for 30 min.

Article Title: Dephosphorylation is the Mechanism of Fibroblast Growth Factor Inhibition of Guanylyl Cyclase-B
Article Snippet: .. Briefly, ~200–500 μg crude membrane protein was diluted to 0.5 or 1 ml in 50 mM Tris-HCl pH 7.5, 50 mM NaF, 10 mM NaH2 PO4 , 2 mM EDTA, 0.5% deoxycholate, 0.1% SDS, 1% NP-40, 100 mM NaCl, 10 mM NaH2 PO4 , 1X Roche Protease Inhibitor Cocktail, and 1 μM microcystin. .. After adding 0.6 or 1 μl anti-GC-B rabbit polyclonal antiserum 6328, made against a C-terminal peptide of GC-B [ ], samples were rotated at 4 °C for 1 hour, then added to 25 or 50 μl Protein A/G magnetic beads (ThermoFisher Scientific) and rotated overnight at 4 °C.

Article Title: A Functional Screen Provides Evidence for a Conserved, Regulatory, Juxtamembrane Phosphorylation Site in Guanylyl Cyclase A and B
Article Snippet: .. Membrane preparation Crude membranes were prepared at 4°C in phosphatase inhibitor buffer which consisted of 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid – pH 7.4, 50 mM NaCl, 20% glycerol, 50 mM NaF, 1 mM EDTA, 0.5 µM microcystin and 1X Roche protease inhibitor cocktail. .. Guanylyl cyclase assays Single substrate concentration GC assays were performed at 37°C for 5 min in a buffer containing 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid – pH 7.4, 50 mM NaCl, 0.1% BSA, 0.5 mM 1-methyl-3-isobutylxanthine, 1 mM GTP, 0.5 µM microcystin, 1 mM EDTA, and 1–2 µCi of [α-32 P] GTP, with either 5 mM MgCl2 , 1 mM ATP, and 1 µM natriuretic peptide or 1% Triton X-100 with 5 mM MnCl2 being substituted for the MgCl2 .

Article Title: A synthetic biology approach identifies the mammalian UPR RNA ligase RtcB
Article Snippet: .. Cell pellets were lysed in a buffer containing 30 mMTris-HCl, pH 7.4, 150 mMNaCl, 2mM MgCl2 , 0.5% Triton X-100, 5 mM β-mercaptoethanol, 1 mM PMSF, 1X Roche protease inhibitor cocktail and 1 X Sigma phosphatase inhibitor cocktail. .. Flag-tagged RtcB proteins were affinity-purified from the lysates with anti-Flag M2 affinity-gel (Sigma) followed by extensive washes.

Article Title: Platinum-based drugs disrupt STAT6-mediated suppression of immune responses against cancer in humans and mice
Article Snippet: .. Regardless of cell type, 1 × 106 cells were lysed in 100 μl phosphatase-inhibiting lysis buffer containing 10 mM Tris/HCl, pH 7.8, 5 mM EDTA, 50 mM NaCl, 1 mM Na3 VO4 , 10 mM pyrophosphate, 50 mM NaF, 1% Triton X-100, 1 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 1X Roche protease inhibitor cocktail (Roche Diagnostics Nederland BV). .. Before polyacrylamide gel electrophoresis, reducing sample buffer (62.5 mM Tris/HCl, pH 6.8, 25% v/v glycerol, 2% w/v sodium dodecyl sulfate, 0.01% w/v bromophenol blue, 5% v/v/β-mercaptoethanol) was added 1:1 to a lysate equivalent of approximately 200,000 cells.

Article Title: Histone deacetylases 1 and 2 maintain S-phase chromatin and DNA replication fork progression
Article Snippet: .. Cell lysis buffer (10 mM Tris–HCl, pH 7.4, 300 mM sucrose, 3 mM CaCl2 , 2 mM Mg(CH3COO)2, 0.5% NP-40, 5 mM dithiothreitol (DTT) and 1X Roche protease inhibitor cocktail) was added to the plate and left on ice for 5 min. .. Cells were scraped following lysis, Dounce homogenized fifty times, spun at 1000 rpm for 5 min at 4°C.

Article Title: RAR/RXR binding dynamics distinguish pluripotency from differentiation associated cis-regulatory elements
Article Snippet: .. Following repeated phosphate-buffered saline (PBS) washing, cell pellets were lysed in FAIRE lysis buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-Cl [pH 8.0], 1 mM EDTA, 1X Roche protease inhibitor cocktail) and sonicated with a Bioruptor (Diagenode) for 15 min at high intensity 30s ON, 30s OFF. ..

Imaging:

Article Title: The chemotherapeutic drug oxaliplatin differentially affects blood DC function dependent on environmental cues
Article Snippet: Preparation of protein lysates and Western blotting 2 × 105 cells were lysed in 20 μl lysis buffer containing 10 mM Tris/HCl pH 7.8, 5 mM EDTA, 50 mM NaCl, 1 mM Na3 VO4 10 mM pyrophosphate, 50 mM NaF, 1% Triton X-100, 1 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin and 1X Roche protease inhibitor cocktail (Roche Diagnostics Nederland BV, Almere, the Netherlands). .. After washing, the membranes were incubated with one of the goat-anti-mouseIRDye800CW (LI-COR Biosciences, Lincoln, NE) or polyclonal goat-anti-rabbitAlexaFluor-680 (Molecular Probes, Eugene, OR) as secondary antibody and analyzed with the LICOR Odyssey Imaging system (LI-COR Biosciences).

Protein Concentration:

Article Title: Modulation of ?-Secretase Activity by Multiple Enzyme-Substrate Interactions: Implications in Pathogenesis of Alzheimer's Disease
Article Snippet: .. The cell pellets were re-suspended in 20 mM Pipes pH = 7.0, 140 mM KCl, 0.25 M sucrose, 5 mM EGTA, plus 1X Roche protease inhibitors cocktail, so that the total protein concentration was 10 mg/ml. ..

Sonication:

Article Title: RAR/RXR binding dynamics distinguish pluripotency from differentiation associated cis-regulatory elements
Article Snippet: .. Following repeated phosphate-buffered saline (PBS) washing, cell pellets were lysed in FAIRE lysis buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-Cl [pH 8.0], 1 mM EDTA, 1X Roche protease inhibitor cocktail) and sonicated with a Bioruptor (Diagenode) for 15 min at high intensity 30s ON, 30s OFF. ..

Affinity Purification:

Article Title: A synthetic biology approach identifies the mammalian UPR RNA ligase RtcB
Article Snippet: Cell pellets were lysed in a buffer containing 30 mMTris-HCl, pH 7.4, 150 mMNaCl, 2mM MgCl2 , 0.5% Triton X-100, 5 mM β-mercaptoethanol, 1 mM PMSF, 1X Roche protease inhibitor cocktail and 1 X Sigma phosphatase inhibitor cocktail. .. Flag-tagged RtcB proteins were affinity-purified from the lysates with anti-Flag M2 affinity-gel (Sigma) followed by extensive washes.

Nucleic Acid Electrophoresis:

Article Title: Dephosphorylation is the Mechanism of Fibroblast Growth Factor Inhibition of Guanylyl Cyclase-B
Article Snippet: Paragraph title: 2.7. Phos-tag gel electrophoresis ... Briefly, ~200–500 μg crude membrane protein was diluted to 0.5 or 1 ml in 50 mM Tris-HCl pH 7.5, 50 mM NaF, 10 mM NaH2 PO4 , 2 mM EDTA, 0.5% deoxycholate, 0.1% SDS, 1% NP-40, 100 mM NaCl, 10 mM NaH2 PO4 , 1X Roche Protease Inhibitor Cocktail, and 1 μM microcystin.

Magnetic Beads:

Article Title: Dephosphorylation is the Mechanism of Fibroblast Growth Factor Inhibition of Guanylyl Cyclase-B
Article Snippet: Briefly, ~200–500 μg crude membrane protein was diluted to 0.5 or 1 ml in 50 mM Tris-HCl pH 7.5, 50 mM NaF, 10 mM NaH2 PO4 , 2 mM EDTA, 0.5% deoxycholate, 0.1% SDS, 1% NP-40, 100 mM NaCl, 10 mM NaH2 PO4 , 1X Roche Protease Inhibitor Cocktail, and 1 μM microcystin. .. After adding 0.6 or 1 μl anti-GC-B rabbit polyclonal antiserum 6328, made against a C-terminal peptide of GC-B [ ], samples were rotated at 4 °C for 1 hour, then added to 25 or 50 μl Protein A/G magnetic beads (ThermoFisher Scientific) and rotated overnight at 4 °C.

Isolation:

Article Title: Histone deacetylases 1 and 2 maintain S-phase chromatin and DNA replication fork progression
Article Snippet: Paragraph title: Nuclei isolation for micrococcal nuclease digestion ... Nuclei were stored in nuclei storage buffer (50 mM Tris- HCl, pH 8.3, 40% glycerol, 0.1 mM EDTA, 5 mM Mg(CH3COO)2, 5 mM DTT and 1X Roche protease inhibitor cocktail) and stored at −80°C until use.

Article Title: RAR/RXR binding dynamics distinguish pluripotency from differentiation associated cis-regulatory elements
Article Snippet: Paragraph title: Formaldehyde assisted isolation of regulatory elements (FAIRE) ... Following repeated phosphate-buffered saline (PBS) washing, cell pellets were lysed in FAIRE lysis buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-Cl [pH 8.0], 1 mM EDTA, 1X Roche protease inhibitor cocktail) and sonicated with a Bioruptor (Diagenode) for 15 min at high intensity 30s ON, 30s OFF.

Labeling:

Article Title: Histone deacetylases 1 and 2 maintain S-phase chromatin and DNA replication fork progression
Article Snippet: Nuclei isolation for micrococcal nuclease digestion NIH3T3 cells were labeled with 20 μM BrdU, washed with ice-cold phosphate-buffered saline (PBS). .. Nuclei were stored in nuclei storage buffer (50 mM Tris- HCl, pH 8.3, 40% glycerol, 0.1 mM EDTA, 5 mM Mg(CH3COO)2, 5 mM DTT and 1X Roche protease inhibitor cocktail) and stored at −80°C until use.

Purification:

Article Title: The chemotherapeutic drug oxaliplatin differentially affects blood DC function dependent on environmental cues
Article Snippet: Preparation of protein lysates and Western blotting 2 × 105 cells were lysed in 20 μl lysis buffer containing 10 mM Tris/HCl pH 7.8, 5 mM EDTA, 50 mM NaCl, 1 mM Na3 VO4 10 mM pyrophosphate, 50 mM NaF, 1% Triton X-100, 1 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin and 1X Roche protease inhibitor cocktail (Roche Diagnostics Nederland BV, Almere, the Netherlands). .. After blocking, membranes were incubated with one the following antibodies: mouse-monoclonal-anti-β-actin (1:20,000; Sigma-Aldrich, St. Louis, MO), purified mouse-anti-PTP1C/Shp-1 (1:250; BD), rabbit-polyclonal-anti-STAT1, rabbit-polyclonal-anti-pSTAT1, rabbit-monoclonal-anti-STAT3 and rabbit-monoclonal-anti-pSTAT3 (all from Cell Signaling).

Article Title: A synthetic biology approach identifies the mammalian UPR RNA ligase RtcB
Article Snippet: Flag-RtcB and Flag-RtcBC122A proteins were purified from stable HEK293T cell lines that expressed the two proteins, respectively. .. Cell pellets were lysed in a buffer containing 30 mMTris-HCl, pH 7.4, 150 mMNaCl, 2mM MgCl2 , 0.5% Triton X-100, 5 mM β-mercaptoethanol, 1 mM PMSF, 1X Roche protease inhibitor cocktail and 1 X Sigma phosphatase inhibitor cocktail.

Polyacrylamide Gel Electrophoresis:

Article Title: Platinum-based drugs disrupt STAT6-mediated suppression of immune responses against cancer in humans and mice
Article Snippet: Regardless of cell type, 1 × 106 cells were lysed in 100 μl phosphatase-inhibiting lysis buffer containing 10 mM Tris/HCl, pH 7.8, 5 mM EDTA, 50 mM NaCl, 1 mM Na3 VO4 , 10 mM pyrophosphate, 50 mM NaF, 1% Triton X-100, 1 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 1X Roche protease inhibitor cocktail (Roche Diagnostics Nederland BV). .. Before polyacrylamide gel electrophoresis, reducing sample buffer (62.5 mM Tris/HCl, pH 6.8, 25% v/v glycerol, 2% w/v sodium dodecyl sulfate, 0.01% w/v bromophenol blue, 5% v/v/β-mercaptoethanol) was added 1:1 to a lysate equivalent of approximately 200,000 cells.

Staining:

Article Title: Dephosphorylation is the Mechanism of Fibroblast Growth Factor Inhibition of Guanylyl Cyclase-B
Article Snippet: Paragraph title: 2.6. Immunoprecipitations and ProQ or SYPRO Ruby Staining ... RCS cells were lysed for 30 min at 4°C on a rotator in RIPA buffer containing: 50 mM HEPES pH 7.4, 50 mM NaF, 2 mM EDTA, 0.5% deoxycholate, 0.1% SDS, 1% IGEPAL CA-630, 100 mM NaCl, 10 mM NaH2 PO4 , 1X Roche Protease Inhibitor Cocktail, and 0.5 μM microcystin.

Plasmid Preparation:

Article Title: A synthetic biology approach identifies the mammalian UPR RNA ligase RtcB
Article Snippet: To reconstitute XBP1u splicing in vitro , we first cloned a cDNA fragment encodingV5-XBP1uin -Rn luciferase into a pBluescript vector downstream of T7 promoter. .. Cell pellets were lysed in a buffer containing 30 mMTris-HCl, pH 7.4, 150 mMNaCl, 2mM MgCl2 , 0.5% Triton X-100, 5 mM β-mercaptoethanol, 1 mM PMSF, 1X Roche protease inhibitor cocktail and 1 X Sigma phosphatase inhibitor cocktail.

Sample Prep:

Article Title: Calcineurin Targets Involved in Stress Survival and Fungal Virulence
Article Snippet: Paragraph title: Sample preparation for Crz1 phosphoproteomics ... The beads were resuspended in ice-cold PBS with 1X Roche protease inhibitor cocktail, and 1 mM PMSF added and submitted to the Duke Proteomic Core Facility for phosphopeptide enrichment and phosphosite determination.

In Vitro:

Article Title: A synthetic biology approach identifies the mammalian UPR RNA ligase RtcB
Article Snippet: Paragraph title: Reconstitution of XBP1u splicing reaction in vitro ... Cell pellets were lysed in a buffer containing 30 mMTris-HCl, pH 7.4, 150 mMNaCl, 2mM MgCl2 , 0.5% Triton X-100, 5 mM β-mercaptoethanol, 1 mM PMSF, 1X Roche protease inhibitor cocktail and 1 X Sigma phosphatase inhibitor cocktail.

Homogenization:

Article Title: Calcineurin Targets Involved in Stress Survival and Fungal Virulence
Article Snippet: Cells were lysed using a mini-bead beater for 10 cycles (90 seconds homogenization with 2 minutes rest intervals) and the cell lysates cleared by centrifugation at 3,000 rpm for 10 minutes, at 4°C. .. The beads were resuspended in ice-cold PBS with 1X Roche protease inhibitor cocktail, and 1 mM PMSF added and submitted to the Duke Proteomic Core Facility for phosphopeptide enrichment and phosphosite determination.

Immunoprecipitation:

Article Title: Dephosphorylation is the Mechanism of Fibroblast Growth Factor Inhibition of Guanylyl Cyclase-B
Article Snippet: For analysis of phosphorylation by Phos-tag, GC-B was immunoprecipitated as previously described [ ]. .. Briefly, ~200–500 μg crude membrane protein was diluted to 0.5 or 1 ml in 50 mM Tris-HCl pH 7.5, 50 mM NaF, 10 mM NaH2 PO4 , 2 mM EDTA, 0.5% deoxycholate, 0.1% SDS, 1% NP-40, 100 mM NaCl, 10 mM NaH2 PO4 , 1X Roche Protease Inhibitor Cocktail, and 1 μM microcystin.

Lysis:

Article Title: Histone deacetylases 1 and 2 maintain S-phase chromatin and DNA replication fork progression
Article Snippet: Cells were scraped following lysis, Dounce homogenized fifty times, spun at 1000 rpm for 5 min at 4°C. .. Nuclei were stored in nuclei storage buffer (50 mM Tris- HCl, pH 8.3, 40% glycerol, 0.1 mM EDTA, 5 mM Mg(CH3COO)2, 5 mM DTT and 1X Roche protease inhibitor cocktail) and stored at −80°C until use.

Article Title: Calcineurin Targets Involved in Stress Survival and Fungal Virulence
Article Snippet: The beads were washed twice with lysis buffer, twice with wash buffer (10 mM Tris/HCl, pH 7.5, 500 mM NaCl, 0.5 mM EDTA), and twice with lysis buffer without the protease inhibitor cocktail. .. The beads were resuspended in ice-cold PBS with 1X Roche protease inhibitor cocktail, and 1 mM PMSF added and submitted to the Duke Proteomic Core Facility for phosphopeptide enrichment and phosphosite determination.

Article Title: The chemotherapeutic drug oxaliplatin differentially affects blood DC function dependent on environmental cues
Article Snippet: .. Preparation of protein lysates and Western blotting 2 × 105 cells were lysed in 20 μl lysis buffer containing 10 mM Tris/HCl pH 7.8, 5 mM EDTA, 50 mM NaCl, 1 mM Na3 VO4 10 mM pyrophosphate, 50 mM NaF, 1% Triton X-100, 1 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin and 1X Roche protease inhibitor cocktail (Roche Diagnostics Nederland BV, Almere, the Netherlands). ..

Article Title: Platinum-based drugs disrupt STAT6-mediated suppression of immune responses against cancer in humans and mice
Article Snippet: .. Regardless of cell type, 1 × 106 cells were lysed in 100 μl phosphatase-inhibiting lysis buffer containing 10 mM Tris/HCl, pH 7.8, 5 mM EDTA, 50 mM NaCl, 1 mM Na3 VO4 , 10 mM pyrophosphate, 50 mM NaF, 1% Triton X-100, 1 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 1X Roche protease inhibitor cocktail (Roche Diagnostics Nederland BV). .. Before polyacrylamide gel electrophoresis, reducing sample buffer (62.5 mM Tris/HCl, pH 6.8, 25% v/v glycerol, 2% w/v sodium dodecyl sulfate, 0.01% w/v bromophenol blue, 5% v/v/β-mercaptoethanol) was added 1:1 to a lysate equivalent of approximately 200,000 cells.

Article Title: Histone deacetylases 1 and 2 maintain S-phase chromatin and DNA replication fork progression
Article Snippet: .. Cell lysis buffer (10 mM Tris–HCl, pH 7.4, 300 mM sucrose, 3 mM CaCl2 , 2 mM Mg(CH3COO)2, 0.5% NP-40, 5 mM dithiothreitol (DTT) and 1X Roche protease inhibitor cocktail) was added to the plate and left on ice for 5 min. .. Cells were scraped following lysis, Dounce homogenized fifty times, spun at 1000 rpm for 5 min at 4°C.

Article Title: RAR/RXR binding dynamics distinguish pluripotency from differentiation associated cis-regulatory elements
Article Snippet: .. Following repeated phosphate-buffered saline (PBS) washing, cell pellets were lysed in FAIRE lysis buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-Cl [pH 8.0], 1 mM EDTA, 1X Roche protease inhibitor cocktail) and sonicated with a Bioruptor (Diagenode) for 15 min at high intensity 30s ON, 30s OFF. ..

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  • 95
    Roche dephosphorylation buffer
    <t>Dephosphorylation</t> of HDAC5 S279 in the NAc limits the development of cocaine reward behavior. A. Representative image of HSV-mediated gene expression in the NAc. GFP expression was used to confirm virus injection placement. B. Western blotting showing
    Dephosphorylation Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 95/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dephosphorylation buffer/product/Roche
    Average 95 stars, based on 24 article reviews
    Price from $9.99 to $1999.99
    dephosphorylation buffer - by Bioz Stars, 2020-04
    95/100 stars
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    80
    Roche triton x100 extraction buffer
    Reduction of heparan sulfate biosynthetic function prevents increases of ubiquitin-modified protein in the brains of ROS exposed animals. Accumulation of insoluble ubiquitinated proteins (IUPs) was examined in the <t>triton-X100</t> insoluble fraction of total head proteins. UAS-mCherry RNAi, an shRNAi with no predicted targets in the Drosophila genome, was used as a control. All plus/minus oxidant pairs are from the same blot. All lanes are from the same experiment, with the same standard sample loaded on all gels from this one experiment for standardization (see Supplemental Figure 3 for original blots). Overexpression of Atg8a ( UAS-Atg8a ) was used as a positive control for enhancement of autophagy. The UAS-constructs were expressed under control of elav -GAL4 with UAS-dcrII to enhance RNAi efficacy. Lanes from anti-ubiquitin stained membranes are shown in control vs. oxidant-exposed pairs. The ratio depicted is the average density of anti-ubiquitin staining, normalized to loading, in samples with peroxide (+) divided by samples without peroxide (-). Two different sfl RNAi lines were tested. Two additional replicate experiments gave average peroxide/no peroxide ratios of 1.23 for control, and 0.8 for sfl RNAi-HMS . Two replicates of ttv RNAi also showed lower levels of ubiquitin-insoluble material upon peroxide exposure (ratio of 0.7).
    Triton X100 Extraction Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche rap1 storage buffer
    <t>Rap1</t> protects the 5′ end when bound to dsDNA 2 nt inwards of the ds-ss junction. ( a,d ) The substrates used for DEPA experiments. The black bar indicates the guide sequence, and “*” the radioactive label (as described in Fig. 2 legend). ( a ) The telomere substrate D17S9 contains 17 bp dsDNA and a 9 nt 3′ overhang. The Rap1 MBS (bold text) is located 2 nt inwards of the ds-ss junction. ( d ) Substrate D17S9m contains 17 bp telomere dsDNA with four mutations in the Rap1 MBS (bold letters, mutations in red) and a 9 nt ssDNA 3′ overhang. ( b,e ) DEPA sequencing gels of D17S9 ( b ) and D17S9m ( e ), showing the loading control (LC) and uncleaved substrate (S) from the Rap1 containing and BSA control reactions at 0; 20; 40; 60; 120 s. Full-size gel showing all the reaction products can be found in Supplementary Fig. S4 . ( c,f ) Quantification of the gels shown in ( b ) and ( e ) respectively.
    Rap1 Storage Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche complete edta free protease inhibitor cocktail
    <t>Rap1</t> protects the 5′ end when bound to dsDNA 2 nt inwards of the ds-ss junction. ( a,d ) The substrates used for DEPA experiments. The black bar indicates the guide sequence, and “*” the radioactive label (as described in Fig. 2 legend). ( a ) The telomere substrate D17S9 contains 17 bp dsDNA and a 9 nt 3′ overhang. The Rap1 MBS (bold text) is located 2 nt inwards of the ds-ss junction. ( d ) Substrate D17S9m contains 17 bp telomere dsDNA with four mutations in the Rap1 MBS (bold letters, mutations in red) and a 9 nt ssDNA 3′ overhang. ( b,e ) DEPA sequencing gels of D17S9 ( b ) and D17S9m ( e ), showing the loading control (LC) and uncleaved substrate (S) from the Rap1 containing and BSA control reactions at 0; 20; 40; 60; 120 s. Full-size gel showing all the reaction products can be found in Supplementary Fig. S4 . ( c,f ) Quantification of the gels shown in ( b ) and ( e ) respectively.
    Complete Edta Free Protease Inhibitor Cocktail, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 372 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Dephosphorylation of HDAC5 S279 in the NAc limits the development of cocaine reward behavior. A. Representative image of HSV-mediated gene expression in the NAc. GFP expression was used to confirm virus injection placement. B. Western blotting showing

    Journal: Neuron

    Article Title: Histone deacetylase 5 limits cocaine reward through cAMP-induced nuclear import

    doi: 10.1016/j.neuron.2011.10.032

    Figure Lengend Snippet: Dephosphorylation of HDAC5 S279 in the NAc limits the development of cocaine reward behavior. A. Representative image of HSV-mediated gene expression in the NAc. GFP expression was used to confirm virus injection placement. B. Western blotting showing

    Article Snippet: IPed HDAC5 from striatal neurons in RIPA buffer was washed with dephosphorylation buffer (50 mM Tris-HCl, pH8.5, 20 mM MgCl2 , 1 mM DTT, protease inhibitor cocktail (1X; Roche)) five times and incubated with or without 2.5 U of purified PP2A (Promega) at 30°C for 60 min. Proteins were subjected to western blotting analysis.

    Techniques: De-Phosphorylation Assay, Expressing, Injection, Western Blot

    cAMP signaling promotes dephosphorylation of S279, a novel Cdk5 phosphorylation site of HDAC5. A. Cultured striatal neurons were treated with vehicle or the Cdk5 inhibitor roscovitine (50 μM) for 3 hrs. IPed HDAC5 from cultured striatal neurons

    Journal: Neuron

    Article Title: Histone deacetylase 5 limits cocaine reward through cAMP-induced nuclear import

    doi: 10.1016/j.neuron.2011.10.032

    Figure Lengend Snippet: cAMP signaling promotes dephosphorylation of S279, a novel Cdk5 phosphorylation site of HDAC5. A. Cultured striatal neurons were treated with vehicle or the Cdk5 inhibitor roscovitine (50 μM) for 3 hrs. IPed HDAC5 from cultured striatal neurons

    Article Snippet: IPed HDAC5 from striatal neurons in RIPA buffer was washed with dephosphorylation buffer (50 mM Tris-HCl, pH8.5, 20 mM MgCl2 , 1 mM DTT, protease inhibitor cocktail (1X; Roche)) five times and incubated with or without 2.5 U of purified PP2A (Promega) at 30°C for 60 min. Proteins were subjected to western blotting analysis.

    Techniques: De-Phosphorylation Assay, Cell Culture

    PP2A activity is required for cAMP-induced dephosphorylation of S279 HDAC5. A. Okadaic acid blocks cAMP-induced dephosphorylation of HDAC5. Cultured striatal neurons were treated with forskolin (10 μM) in the presence or absence of okadaic acid

    Journal: Neuron

    Article Title: Histone deacetylase 5 limits cocaine reward through cAMP-induced nuclear import

    doi: 10.1016/j.neuron.2011.10.032

    Figure Lengend Snippet: PP2A activity is required for cAMP-induced dephosphorylation of S279 HDAC5. A. Okadaic acid blocks cAMP-induced dephosphorylation of HDAC5. Cultured striatal neurons were treated with forskolin (10 μM) in the presence or absence of okadaic acid

    Article Snippet: IPed HDAC5 from striatal neurons in RIPA buffer was washed with dephosphorylation buffer (50 mM Tris-HCl, pH8.5, 20 mM MgCl2 , 1 mM DTT, protease inhibitor cocktail (1X; Roche)) five times and incubated with or without 2.5 U of purified PP2A (Promega) at 30°C for 60 min. Proteins were subjected to western blotting analysis.

    Techniques: Activity Assay, De-Phosphorylation Assay, Cell Culture

    Dephosphorylation of S279 is necessary for cAMP-induced nuclear import of HDAC5. A. Okadaic acid prevents cAMP-induced nuclear import of HDAC5. Transfected neurons were treated with vehicle or forskolin (10 μM) in the absence or presence of okadaic

    Journal: Neuron

    Article Title: Histone deacetylase 5 limits cocaine reward through cAMP-induced nuclear import

    doi: 10.1016/j.neuron.2011.10.032

    Figure Lengend Snippet: Dephosphorylation of S279 is necessary for cAMP-induced nuclear import of HDAC5. A. Okadaic acid prevents cAMP-induced nuclear import of HDAC5. Transfected neurons were treated with vehicle or forskolin (10 μM) in the absence or presence of okadaic

    Article Snippet: IPed HDAC5 from striatal neurons in RIPA buffer was washed with dephosphorylation buffer (50 mM Tris-HCl, pH8.5, 20 mM MgCl2 , 1 mM DTT, protease inhibitor cocktail (1X; Roche)) five times and incubated with or without 2.5 U of purified PP2A (Promega) at 30°C for 60 min. Proteins were subjected to western blotting analysis.

    Techniques: De-Phosphorylation Assay, Transfection

    Reduction of heparan sulfate biosynthetic function prevents increases of ubiquitin-modified protein in the brains of ROS exposed animals. Accumulation of insoluble ubiquitinated proteins (IUPs) was examined in the triton-X100 insoluble fraction of total head proteins. UAS-mCherry RNAi, an shRNAi with no predicted targets in the Drosophila genome, was used as a control. All plus/minus oxidant pairs are from the same blot. All lanes are from the same experiment, with the same standard sample loaded on all gels from this one experiment for standardization (see Supplemental Figure 3 for original blots). Overexpression of Atg8a ( UAS-Atg8a ) was used as a positive control for enhancement of autophagy. The UAS-constructs were expressed under control of elav -GAL4 with UAS-dcrII to enhance RNAi efficacy. Lanes from anti-ubiquitin stained membranes are shown in control vs. oxidant-exposed pairs. The ratio depicted is the average density of anti-ubiquitin staining, normalized to loading, in samples with peroxide (+) divided by samples without peroxide (-). Two different sfl RNAi lines were tested. Two additional replicate experiments gave average peroxide/no peroxide ratios of 1.23 for control, and 0.8 for sfl RNAi-HMS . Two replicates of ttv RNAi also showed lower levels of ubiquitin-insoluble material upon peroxide exposure (ratio of 0.7).

    Journal: G3: Genes|Genomes|Genetics

    Article Title: Heparan Sulfate Structure Affects Autophagy, Lifespan, Responses to Oxidative Stress, and Cell Degeneration in Drosophila parkin Mutants

    doi: 10.1534/g3.119.400730

    Figure Lengend Snippet: Reduction of heparan sulfate biosynthetic function prevents increases of ubiquitin-modified protein in the brains of ROS exposed animals. Accumulation of insoluble ubiquitinated proteins (IUPs) was examined in the triton-X100 insoluble fraction of total head proteins. UAS-mCherry RNAi, an shRNAi with no predicted targets in the Drosophila genome, was used as a control. All plus/minus oxidant pairs are from the same blot. All lanes are from the same experiment, with the same standard sample loaded on all gels from this one experiment for standardization (see Supplemental Figure 3 for original blots). Overexpression of Atg8a ( UAS-Atg8a ) was used as a positive control for enhancement of autophagy. The UAS-constructs were expressed under control of elav -GAL4 with UAS-dcrII to enhance RNAi efficacy. Lanes from anti-ubiquitin stained membranes are shown in control vs. oxidant-exposed pairs. The ratio depicted is the average density of anti-ubiquitin staining, normalized to loading, in samples with peroxide (+) divided by samples without peroxide (-). Two different sfl RNAi lines were tested. Two additional replicate experiments gave average peroxide/no peroxide ratios of 1.23 for control, and 0.8 for sfl RNAi-HMS . Two replicates of ttv RNAi also showed lower levels of ubiquitin-insoluble material upon peroxide exposure (ratio of 0.7).

    Article Snippet: Typically, 30-60 adult heads were homogenized by ceramic bead agitation using the Bead Ruptor 24 in 150ul of Triton-X100 extraction buffer (1% Triton-X100, 1x PBS, 1X protease inhibitor cocktail Complete [Roche, 10184600]).

    Techniques: Modification, Over Expression, Positive Control, Construct, Staining

    Rap1 protects the 5′ end when bound to dsDNA 2 nt inwards of the ds-ss junction. ( a,d ) The substrates used for DEPA experiments. The black bar indicates the guide sequence, and “*” the radioactive label (as described in Fig. 2 legend). ( a ) The telomere substrate D17S9 contains 17 bp dsDNA and a 9 nt 3′ overhang. The Rap1 MBS (bold text) is located 2 nt inwards of the ds-ss junction. ( d ) Substrate D17S9m contains 17 bp telomere dsDNA with four mutations in the Rap1 MBS (bold letters, mutations in red) and a 9 nt ssDNA 3′ overhang. ( b,e ) DEPA sequencing gels of D17S9 ( b ) and D17S9m ( e ), showing the loading control (LC) and uncleaved substrate (S) from the Rap1 containing and BSA control reactions at 0; 20; 40; 60; 120 s. Full-size gel showing all the reaction products can be found in Supplementary Fig. S4 . ( c,f ) Quantification of the gels shown in ( b ) and ( e ) respectively.

    Journal: Scientific Reports

    Article Title: Rap1 and Cdc13 have complementary roles in preventing exonucleolytic degradation of telomere 5′ ends

    doi: 10.1038/s41598-017-08663-x

    Figure Lengend Snippet: Rap1 protects the 5′ end when bound to dsDNA 2 nt inwards of the ds-ss junction. ( a,d ) The substrates used for DEPA experiments. The black bar indicates the guide sequence, and “*” the radioactive label (as described in Fig. 2 legend). ( a ) The telomere substrate D17S9 contains 17 bp dsDNA and a 9 nt 3′ overhang. The Rap1 MBS (bold text) is located 2 nt inwards of the ds-ss junction. ( d ) Substrate D17S9m contains 17 bp telomere dsDNA with four mutations in the Rap1 MBS (bold letters, mutations in red) and a 9 nt ssDNA 3′ overhang. ( b,e ) DEPA sequencing gels of D17S9 ( b ) and D17S9m ( e ), showing the loading control (LC) and uncleaved substrate (S) from the Rap1 containing and BSA control reactions at 0; 20; 40; 60; 120 s. Full-size gel showing all the reaction products can be found in Supplementary Fig. S4 . ( c,f ) Quantification of the gels shown in ( b ) and ( e ) respectively.

    Article Snippet: The peak fractions containing Rap1-FL protein were pooled and buffer exchanged against Rap1 storage buffer (20 mM HEPES, pH 8.0, 150 mM NaCl, 1 mM DTT, 10% glycerol, 1x protease inhibitor cocktail (Roche)) on PD-10 column (GE Healthcare) and stored at −80 °C until use.

    Techniques: Sequencing

    Schematic figure summarizing the results of this work and how it is proposed to relate to different in vivo situations. ( a ) Shows the different substrate tested with Cdc13 or Rap1 pre-bound at their respective MBS at various distances relative the ds-ss junction. “ + ” indicates protection, while “−’’ indicates no protection. ( b ) Protection by Rap1 when the 3′ overhang is very short and unable to accommodate Cdc13 binding. ( c ) Protection by Rap1 in a hypothetical situation where Cdc13 is bound very far away from the ds-ss junction (longer than tested here). ( d ) Protection may be provided by Cdc13 alone when the 3′ overhang accommodates its binding. ( e ) The wild type Rap1 DBD 337–582 is firmly attached to its MBS, and fully protects the 5′ end from degradation by λ-exonuclease. ( f ) The Rap1 wrapping loop mutant DBD 337–556 is only partly attached to the MBS, leaving the 5′ end accessible to λ-exonuclease, which cleaves off the first 3 nt of DNA before being halted at a site where the mutant DBD is more firmly attached.

    Journal: Scientific Reports

    Article Title: Rap1 and Cdc13 have complementary roles in preventing exonucleolytic degradation of telomere 5′ ends

    doi: 10.1038/s41598-017-08663-x

    Figure Lengend Snippet: Schematic figure summarizing the results of this work and how it is proposed to relate to different in vivo situations. ( a ) Shows the different substrate tested with Cdc13 or Rap1 pre-bound at their respective MBS at various distances relative the ds-ss junction. “ + ” indicates protection, while “−’’ indicates no protection. ( b ) Protection by Rap1 when the 3′ overhang is very short and unable to accommodate Cdc13 binding. ( c ) Protection by Rap1 in a hypothetical situation where Cdc13 is bound very far away from the ds-ss junction (longer than tested here). ( d ) Protection may be provided by Cdc13 alone when the 3′ overhang accommodates its binding. ( e ) The wild type Rap1 DBD 337–582 is firmly attached to its MBS, and fully protects the 5′ end from degradation by λ-exonuclease. ( f ) The Rap1 wrapping loop mutant DBD 337–556 is only partly attached to the MBS, leaving the 5′ end accessible to λ-exonuclease, which cleaves off the first 3 nt of DNA before being halted at a site where the mutant DBD is more firmly attached.

    Article Snippet: The peak fractions containing Rap1-FL protein were pooled and buffer exchanged against Rap1 storage buffer (20 mM HEPES, pH 8.0, 150 mM NaCl, 1 mM DTT, 10% glycerol, 1x protease inhibitor cocktail (Roche)) on PD-10 column (GE Healthcare) and stored at −80 °C until use.

    Techniques: In Vivo, Binding Assay, Mutagenesis

    Rap1 protects the 5′ end when its binding site contains 2 nt but not 4 nt of ssDNA. ( a ) Telomere part of D13S13 contains 13 bp dsDNA and a 13 nt 3′ overhang. The Rap1 MBS (bold text) is located across the ds-ss junction and contains 2 nt of ssDNA. ( b ) D11S15 telomere part has 11 bp dsDNA and a 15 nt 3′ overhang. The Rap1 MBS locates across the ds-ss junction and contains 4 nt of ssDNA. The black bar indicates the guide sequence, and “*” the radioactive label (as described in Fig. 2 legend). ( c, d ) DEPA gels showing the results from D13S13 ( c ) and D11S15 ( d ). Both ( c ) and ( d ) show the loading control (LC) and uncleaved substrate (S) from the Rap1 containing and BSA control reactions at 0; 20; 40; 60; 120 s. Uncropped gels can be found in Supplementary Fig. S4 . ( e,f ) Quantification of gels shown in ( c ) and ( d ) respectively.

    Journal: Scientific Reports

    Article Title: Rap1 and Cdc13 have complementary roles in preventing exonucleolytic degradation of telomere 5′ ends

    doi: 10.1038/s41598-017-08663-x

    Figure Lengend Snippet: Rap1 protects the 5′ end when its binding site contains 2 nt but not 4 nt of ssDNA. ( a ) Telomere part of D13S13 contains 13 bp dsDNA and a 13 nt 3′ overhang. The Rap1 MBS (bold text) is located across the ds-ss junction and contains 2 nt of ssDNA. ( b ) D11S15 telomere part has 11 bp dsDNA and a 15 nt 3′ overhang. The Rap1 MBS locates across the ds-ss junction and contains 4 nt of ssDNA. The black bar indicates the guide sequence, and “*” the radioactive label (as described in Fig. 2 legend). ( c, d ) DEPA gels showing the results from D13S13 ( c ) and D11S15 ( d ). Both ( c ) and ( d ) show the loading control (LC) and uncleaved substrate (S) from the Rap1 containing and BSA control reactions at 0; 20; 40; 60; 120 s. Uncropped gels can be found in Supplementary Fig. S4 . ( e,f ) Quantification of gels shown in ( c ) and ( d ) respectively.

    Article Snippet: The peak fractions containing Rap1-FL protein were pooled and buffer exchanged against Rap1 storage buffer (20 mM HEPES, pH 8.0, 150 mM NaCl, 1 mM DTT, 10% glycerol, 1x protease inhibitor cocktail (Roche)) on PD-10 column (GE Healthcare) and stored at −80 °C until use.

    Techniques: Binding Assay, Sequencing

    Graph summarizing the results from DEPA experiments assessing Rap1 5′ end protection of the indicated telomere substrates. Each point is the average and error bars ± SEM of three experiments.

    Journal: Scientific Reports

    Article Title: Rap1 and Cdc13 have complementary roles in preventing exonucleolytic degradation of telomere 5′ ends

    doi: 10.1038/s41598-017-08663-x

    Figure Lengend Snippet: Graph summarizing the results from DEPA experiments assessing Rap1 5′ end protection of the indicated telomere substrates. Each point is the average and error bars ± SEM of three experiments.

    Article Snippet: The peak fractions containing Rap1-FL protein were pooled and buffer exchanged against Rap1 storage buffer (20 mM HEPES, pH 8.0, 150 mM NaCl, 1 mM DTT, 10% glycerol, 1x protease inhibitor cocktail (Roche)) on PD-10 column (GE Healthcare) and stored at −80 °C until use.

    Techniques:

    Design of N. castelli Rap1 DBD wrapping loop and latch mutants, and analysis of their DNA binding activities. ( a ) Sequence alignment (determined using uniprot CLUSTAL O(1.2.2) multiple sequence alignment tool) of the S. cer and N. cas wrapping loop. Amino acids of the S. cer sequence known to take part in DNA-interactions are shown in bold black text and amino acids interacting with the Myb1 domain are shown in blue. “*” Identical, “ : ” strongly similar, and “.” weakly similar amino acids. The numbers of the first and last amino acids in the wrapping loop are indicated. The last amino acid of each DBD variant is indicated by arrows; orange for “wrapping loop mutant” DBD 337–556 , green for “latch mutant” DBD 337–572 , red for “wild type” DBD 337–582 . ( b ) Schematic of the generated N. cas DBD deletion variants, containing two Myb-like domains (Myb1 and Myb2), separated by a linker region, varying lengths of the wrapping loop (WL), and a poly-histidine affinity tag (6ˣH). ( c ) EMSA with D17S9, using, 0.8; 0.4; 0.2; 0.1 μg of the indicated DBD. “-’’ no protein added ( d ) Quantification showing the relative amount of probe (in %) found in the lower shifted band in each lane of the gel in ( d ).

    Journal: Scientific Reports

    Article Title: Rap1 and Cdc13 have complementary roles in preventing exonucleolytic degradation of telomere 5′ ends

    doi: 10.1038/s41598-017-08663-x

    Figure Lengend Snippet: Design of N. castelli Rap1 DBD wrapping loop and latch mutants, and analysis of their DNA binding activities. ( a ) Sequence alignment (determined using uniprot CLUSTAL O(1.2.2) multiple sequence alignment tool) of the S. cer and N. cas wrapping loop. Amino acids of the S. cer sequence known to take part in DNA-interactions are shown in bold black text and amino acids interacting with the Myb1 domain are shown in blue. “*” Identical, “ : ” strongly similar, and “.” weakly similar amino acids. The numbers of the first and last amino acids in the wrapping loop are indicated. The last amino acid of each DBD variant is indicated by arrows; orange for “wrapping loop mutant” DBD 337–556 , green for “latch mutant” DBD 337–572 , red for “wild type” DBD 337–582 . ( b ) Schematic of the generated N. cas DBD deletion variants, containing two Myb-like domains (Myb1 and Myb2), separated by a linker region, varying lengths of the wrapping loop (WL), and a poly-histidine affinity tag (6ˣH). ( c ) EMSA with D17S9, using, 0.8; 0.4; 0.2; 0.1 μg of the indicated DBD. “-’’ no protein added ( d ) Quantification showing the relative amount of probe (in %) found in the lower shifted band in each lane of the gel in ( d ).

    Article Snippet: The peak fractions containing Rap1-FL protein were pooled and buffer exchanged against Rap1 storage buffer (20 mM HEPES, pH 8.0, 150 mM NaCl, 1 mM DTT, 10% glycerol, 1x protease inhibitor cocktail (Roche)) on PD-10 column (GE Healthcare) and stored at −80 °C until use.

    Techniques: Binding Assay, Sequencing, Variant Assay, Mutagenesis, Generated

    The Rap1 DBD is sufficient and the wrapping loop is essential for mediating 5′ end protection. ( a ) DEPA on D17S9 was performed after pre-incubation with BSA (0.8 μg), Rap1 DBD 337–582 (0.8 μg), DBD 337–572 (0.4 μg) or DBD 337–582 (0.8 μg), for 0; 20; 40; 60; 120; 240 s. The nucleotide sequence representing each band and its position relative to the Rap1 MBS is shown to the right. The arrow points out the position of the stalling product. LC indicates the loading control. Lane number is below the gel. ( b ) The results shown in ( a ) were quantified and the % of uncleaved full length substrate at each time point was calculated.

    Journal: Scientific Reports

    Article Title: Rap1 and Cdc13 have complementary roles in preventing exonucleolytic degradation of telomere 5′ ends

    doi: 10.1038/s41598-017-08663-x

    Figure Lengend Snippet: The Rap1 DBD is sufficient and the wrapping loop is essential for mediating 5′ end protection. ( a ) DEPA on D17S9 was performed after pre-incubation with BSA (0.8 μg), Rap1 DBD 337–582 (0.8 μg), DBD 337–572 (0.4 μg) or DBD 337–582 (0.8 μg), for 0; 20; 40; 60; 120; 240 s. The nucleotide sequence representing each band and its position relative to the Rap1 MBS is shown to the right. The arrow points out the position of the stalling product. LC indicates the loading control. Lane number is below the gel. ( b ) The results shown in ( a ) were quantified and the % of uncleaved full length substrate at each time point was calculated.

    Article Snippet: The peak fractions containing Rap1-FL protein were pooled and buffer exchanged against Rap1 storage buffer (20 mM HEPES, pH 8.0, 150 mM NaCl, 1 mM DTT, 10% glycerol, 1x protease inhibitor cocktail (Roche)) on PD-10 column (GE Healthcare) and stored at −80 °C until use.

    Techniques: Incubation, Sequencing