Structured Review

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Edta Free, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 72 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 99 stars, based on 72 article reviews
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Clone Assay:

Article Title: Insights into Herpesvirus Tegument Organization from Structural Analyses of the 970 Central Residues of HSV-1 UL36 Protein
Article Snippet: Paragraph title: Cloning, Expression, and Purification of UL36 Fragments ... The pellet was resuspended in 50 m m HEPES, pH 7.0, 500 m m NaCl, 10% glycerol, 1 m m DTT supplemented by a tablet of EDTA-free “Complete” protease inhibitor mixture tablet (Roche Diagnostics) and lysed by sonication at 4 °C.

Centrifugation:

Article Title: Effect of MyBP-C Binding to Actin on Contractility in Heart Muscle
Article Snippet: .. Cells, collected by centrifugation, were lysed in 50 ml of B-PER bacterial protein extraction reagent (Pierce Chemical Co.), with the addition of 0.3 M NaCl, 5 mM 2-mercaptoethanol and “complete, EDTA-free” protease inhibitor cocktail tablets (Roche Molecular Biochemicals). .. Lysates were sonicated with three 30-s bursts on a Branson Sonifier 450 (Branson Ultrasonics) and clarified by spinning at 20,000 rpm for 15 min For the first metal affinity purification, 5 ml Ni-NTA resin (QIAGEN) was equilibrated in “binding buffer,” containing 25 mM phosphate, 20 mM Tris-Cl, 10% glycerol, 0.3 M KCl, 5 mM 2-mercaptoethanol, and 0.5% Triton X-100, pH 8.0.

Article Title: Insights into Herpesvirus Tegument Organization from Structural Analyses of the 970 Central Residues of HSV-1 UL36 Protein
Article Snippet: Cells expressing construct 760–1733 were pelleted by centrifugation at 5000 × g for 20 min in a JLA9.1,000 rotor (Beckman Coulter). .. The pellet was resuspended in 50 m m HEPES, pH 7.0, 500 m m NaCl, 10% glycerol, 1 m m DTT supplemented by a tablet of EDTA-free “Complete” protease inhibitor mixture tablet (Roche Diagnostics) and lysed by sonication at 4 °C.

Article Title: Thermodynamic Protein Destabilization by GFP Tagging: A Case of Interdomain Allostery
Article Snippet: Purification of eGFP was carried out by resuspending the cells in 50 mM Tris buffer (pH 7.5) containing 100 mM NaCl, 10 mM imidazole, 0.1 mM DTT (buffer B), and the cOmplete, EDTA-free, inhibitor cocktail (Roche Applied Science). .. The cells were then disrupted using a French press and sonication and the lysate was clarified by centrifugation.

Article Title: Intrabodies binding the proline-rich domains of mutant huntingtin increase its turnover and reduce neurotoxicity
Article Snippet: .. Cells were dislodged by mechanical dissociation and pipetting 48 hours post-transfection, harvested by centrifugation, washed with PBS and lysed by sonication in 500 μl lysis buffer (25 mM Hepes, 50 mM NaCl, 1 mM MgCl2 , 0.5 % triton) containing 1 Complete, Mini, EDTA-free; Protease Inhibitor Cocktail tablet (Roche) per 7 ml buffer. .. The soluble protein fraction was collected by centrifugation for 20 min at 4° C at 20,000x g. The insoluble pellet was sonicated in 150 μl 6 M urea and incubated for 20 min at RT.

Expressing:

Article Title: Effect of MyBP-C Binding to Actin on Contractility in Heart Muscle
Article Snippet: Paragraph title: Expression of Recombinant C0C2 ... Cells, collected by centrifugation, were lysed in 50 ml of B-PER bacterial protein extraction reagent (Pierce Chemical Co.), with the addition of 0.3 M NaCl, 5 mM 2-mercaptoethanol and “complete, EDTA-free” protease inhibitor cocktail tablets (Roche Molecular Biochemicals).

Article Title: A Disintegrin-Like and Metalloprotease Domain Containing Thrombospondin Type 1 Motif-like 5 (ADAMTSL5) is a novel fibrillin-1-, fibrillin-2-, and heparin-binding member of the ADAMTS superfamily containing a netrin-like module
Article Snippet: Conditioned 293 SFM II medium was collected from HEK293F cells stably expressing myc-His6 tagged ADAMTSL5 and cultured at 37 °C in 8% CO2 . .. The beads were washed thrice with 0.1% Triton X-100 in PBS, and mixed with 200 μL of 5 μg/mL His6 tagged fibrillin-1 polypeptides rFBN1-N or rFBN1-C representing the N- and C-terminal halves of human fibrillin-1, in Tris-buffered saline with 0.1% Triton X-100 containing EDTA-free, complete mini-protease inhibitor cocktail (Roche) at RT for three hours.

Article Title: Insights into Herpesvirus Tegument Organization from Structural Analyses of the 970 Central Residues of HSV-1 UL36 Protein
Article Snippet: Paragraph title: Cloning, Expression, and Purification of UL36 Fragments ... The pellet was resuspended in 50 m m HEPES, pH 7.0, 500 m m NaCl, 10% glycerol, 1 m m DTT supplemented by a tablet of EDTA-free “Complete” protease inhibitor mixture tablet (Roche Diagnostics) and lysed by sonication at 4 °C.

Article Title: Thermodynamic Protein Destabilization by GFP Tagging: A Case of Interdomain Allostery
Article Snippet: Paragraph title: Expression and purification of eGFP ... Purification of eGFP was carried out by resuspending the cells in 50 mM Tris buffer (pH 7.5) containing 100 mM NaCl, 10 mM imidazole, 0.1 mM DTT (buffer B), and the cOmplete, EDTA-free, inhibitor cocktail (Roche Applied Science).

Stable Transfection:

Article Title: A Disintegrin-Like and Metalloprotease Domain Containing Thrombospondin Type 1 Motif-like 5 (ADAMTSL5) is a novel fibrillin-1-, fibrillin-2-, and heparin-binding member of the ADAMTS superfamily containing a netrin-like module
Article Snippet: Conditioned 293 SFM II medium was collected from HEK293F cells stably expressing myc-His6 tagged ADAMTSL5 and cultured at 37 °C in 8% CO2 . .. The beads were washed thrice with 0.1% Triton X-100 in PBS, and mixed with 200 μL of 5 μg/mL His6 tagged fibrillin-1 polypeptides rFBN1-N or rFBN1-C representing the N- and C-terminal halves of human fibrillin-1, in Tris-buffered saline with 0.1% Triton X-100 containing EDTA-free, complete mini-protease inhibitor cocktail (Roche) at RT for three hours.

Construct:

Article Title: Insights into Herpesvirus Tegument Organization from Structural Analyses of the 970 Central Residues of HSV-1 UL36 Protein
Article Snippet: Cells expressing construct 760–1733 were pelleted by centrifugation at 5000 × g for 20 min in a JLA9.1,000 rotor (Beckman Coulter). .. The pellet was resuspended in 50 m m HEPES, pH 7.0, 500 m m NaCl, 10% glycerol, 1 m m DTT supplemented by a tablet of EDTA-free “Complete” protease inhibitor mixture tablet (Roche Diagnostics) and lysed by sonication at 4 °C.

Electrophoresis:

Article Title: Complement factor H protects mice from ischemic acute kidney injury but is not critical for controlling complement activation by glomerular IgM
Article Snippet: Plasma samples were diluted 1:10 in PBS and reduced by boiling with 0.15 M dithiothreitol for 10 min. at 100° C. Renal tissue was homogenized in RIPA lysis buffer containing 1% Triton X-100, 0.5% deoxycholic acid, 150 mM NaCl, 20 mM β-glycerophosphate, 20 mM Tris·HCl (pH 8.0), 5 mM EGTA, 3 mM MgCl2 , 0.1% SDS, 1mM DTT, 50μM Na3 VO4 , and one tablet of complete, EDTA-free, protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN). .. The proteins were resolved by electrophoresis with a 10% Tris-HCl gel (Bio-Rad, Hercules, CA), and transferred to a polyvinylidene fluoride membrane.

Quantitation Assay:

Article Title: Acetate Availability and Utilization Supports the Growth of Mutant Sub-Populations on Aging Bacterial Colonies
Article Snippet: The cells were pelleted and lysed by the addition of 400 µl lysis buffer (20 mM HEPES pH 8.0, 9 M urea) containing a protease inhibitor cocktail added according to the manufacturers instructions (Complete, Mini, EDTA-free, Roche Diagnostics Scandinavia AB, Bromma, Sweden) and mixed by pipetting. .. An LTQ-Orbitrap Velos Pro ETD mass spectrometer (Thermo Fisher Scientific) was used for the mass spectrometry measurements and the PinPoint 1.3 software was used for the quantitation analysis .

Mass Spectrometry:

Article Title: Acetate Availability and Utilization Supports the Growth of Mutant Sub-Populations on Aging Bacterial Colonies
Article Snippet: Paragraph title: Protein preparation and mass spectrometry measurements ... The cells were pelleted and lysed by the addition of 400 µl lysis buffer (20 mM HEPES pH 8.0, 9 M urea) containing a protease inhibitor cocktail added according to the manufacturers instructions (Complete, Mini, EDTA-free, Roche Diagnostics Scandinavia AB, Bromma, Sweden) and mixed by pipetting.

BIA-KA:

Article Title: MicroRNA‐containing extracellular vesicles released from endothelial colony‐forming cells modulate angiogenesis during ischaemic retinopathy
Article Snippet: Protein extraction EV protein was extracted using radioimmunoprecipitation assay (RIPA) buffer supplemented with cOmplete Mini, EDTA‐free, Protease Inhibitor Cocktail (Roche, Burgess Hill, UK). .. Quantification was performed using a Micro BCA™ Protein Assay Reagent Kit (Thermo Fisher Scientific).

Article Title: Proteomic Analysis of Detergent Resistant Membrane Domains during Early Interaction of Macrophages with Rough and Smooth Brucella melitensis
Article Snippet: Protein concentrations were determined using the BCA protein assay performed in microtiter plates (PIERCE). .. The protease inhibitor cocktail Set III, EDTA-free was from Roche.

Modification:

Article Title: JAGGED Controls Growth Anisotropy and Coordination between Cell Size and Cell Cycle during Plant Organogenesis
Article Snippet: .. Chromatin Immunoprecipitation JAG-GR was activated as described above and ChIP was as described [ ], except for the following modifications: 70–80 inflorescence apices were used per sample; fixation buffer was modified to pH 8.5 and phenylmethylsulfonyl fluoride (PMSF) used at 0.1 mM; after grinding in liquid nitrogen, 300–500 mg tissue powder was resuspended in 700 μl of lysis buffer, modified to contain 0.1 mM PMSF and two tablets of protease inhibitor cocktail complete Mini, EDTA-free (Roche) added per 50 ml of buffer; the supernatant after sonication was precleared for 2 hr with 25 μl Dynabeads Protein A beads (Invitrogen) equilibrated in lysis buffer with 1 mg/ml BSA and 20 μg/ml sonicated salmon sperm, then incubated with 2 μl GR-antibodies (AB3580, Abcam) per 100 μl lysate at 4°C, overnight; 15 μl of equilibrated Dynabeads Protein A beads were added per 100 μl lysate and incubated at 4°C for 4 hr, before proceeding with washes and decrosslinking as described [ ]. ..

Western Blot:

Article Title: WT1-AS promotes cell apoptosis in hepatocellular carcinoma through down-regulating of WT1
Article Snippet: .. Protein analysis For Western-blots, total proteins were extracted from tissues or cultured cells using RIPA buffer containing protease inhibitors cOmplete, ULTRA, Mini, EDTA-free, EASYpack (Roche, Basel, Switzerland), while the membrane proteins were extracted from tissues by Mem-PER Eukaryotic Membrane Protein Extraction Reagent Kit (Thermo Scientific, Rockford, USA). ..

Article Title: A Disintegrin-Like and Metalloprotease Domain Containing Thrombospondin Type 1 Motif-like 5 (ADAMTSL5) is a novel fibrillin-1-, fibrillin-2-, and heparin-binding member of the ADAMTS superfamily containing a netrin-like module
Article Snippet: The beads were washed thrice with 0.1% Triton X-100 in PBS, and mixed with 200 μL of 5 μg/mL His6 tagged fibrillin-1 polypeptides rFBN1-N or rFBN1-C representing the N- and C-terminal halves of human fibrillin-1, in Tris-buffered saline with 0.1% Triton X-100 containing EDTA-free, complete mini-protease inhibitor cocktail (Roche) at RT for three hours. .. The samples were analyzed by 7.5% SDS-PAGE, and western blotting using monoclonal anti-his antibody (R & D) to detect both ADAMTSL5 and fibrillin-1.

Article Title: Complement factor H protects mice from ischemic acute kidney injury but is not critical for controlling complement activation by glomerular IgM
Article Snippet: Paragraph title: Western blot analysis ... Plasma samples were diluted 1:10 in PBS and reduced by boiling with 0.15 M dithiothreitol for 10 min. at 100° C. Renal tissue was homogenized in RIPA lysis buffer containing 1% Triton X-100, 0.5% deoxycholic acid, 150 mM NaCl, 20 mM β-glycerophosphate, 20 mM Tris·HCl (pH 8.0), 5 mM EGTA, 3 mM MgCl2 , 0.1% SDS, 1mM DTT, 50μM Na3 VO4 , and one tablet of complete, EDTA-free, protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN).

Article Title: Proteomic Analysis of Detergent Resistant Membrane Domains during Early Interaction of Macrophages with Rough and Smooth Brucella melitensis
Article Snippet: The protease inhibitor cocktail Set III, EDTA-free was from Roche. .. SuperSignal West Dura Extended Duration Substrate for the development of Western Blots was from PIERCE.

Article Title: Intrabodies binding the proline-rich domains of mutant huntingtin increase its turnover and reduce neurotoxicity
Article Snippet: Cells were dislodged by mechanical dissociation and pipetting 48 hours post-transfection, harvested by centrifugation, washed with PBS and lysed by sonication in 500 μl lysis buffer (25 mM Hepes, 50 mM NaCl, 1 mM MgCl2 , 0.5 % triton) containing 1 Complete, Mini, EDTA-free; Protease Inhibitor Cocktail tablet (Roche) per 7 ml buffer. .. Immunoblots were then performed using rabbit anti-GFP (1:1000 Molecular Probes, Carlsbad CA.) as primary antibody and HRP-conjugated, goat anti-rabbit (1:10,000 Santa Cruz Biotechnology, Santa Cruz, CA.) as secondary antibody to detect HDx-1-GFP.

Chromatography:

Article Title: Analysis of Histones and Chromatin in Xenopus laevis Egg and Oocyte Extracts
Article Snippet: .. 10× ELB salt solution: 25 mM MgCl2 , 500 mM KCl, 100 mM Hepes-KOH pH 7.7 Dithiothreitol (DTT, 1 M stock in dH2 O, store at −20 °C) Heparin Sepharose CL-6B, GE Healthcare 17-0467-01 Heparin binding buffer: 1× ELB salts, 10% glycerol, 1 mM EDTA, 1 mM DTT Heparin wash buffer: 1× ELB salts, 0.5 M NaCl 10% glycerol, 1 mM EDTA, 1 mM DTT Heparin elution buffer: 1× ELB salts, 2 M NaCl 10% glycerol, 1 mM EDTA, 1 mM DTT Complete protease inhibitors, EDTA-free, Roche 11 873 850 001 PhosSTOP phostphatase inhibitors, Roche 04 906 837 001 Butyric acid, Aldrich B103500 β-glycerophosphate, Calbiochem 35675 Sodium Fluoride, Fisher S299 Phenylmethanesulphonyl fluoride (PMSF, 100 mM stock in isopropanol), Sigma P7626 Econo-Pac chromatography columns, Bio-Rad #732-1010 β-mercaptoethanol (βME), Fisher 100% trichloroacetic acid (TCA), Fisher A322 Oakridge centrifuge tubes, Nalgene #3119-0050 Refrigerated centrifuge (Sorvall SS-34 rotor) Coomassie Brilliant Blue stain: 40% methanol, 10% acetic acid, 0.025% Coomassie R-250 in dH2 O ..

Protease Inhibitor:

Article Title: Effect of MyBP-C Binding to Actin on Contractility in Heart Muscle
Article Snippet: .. Cells, collected by centrifugation, were lysed in 50 ml of B-PER bacterial protein extraction reagent (Pierce Chemical Co.), with the addition of 0.3 M NaCl, 5 mM 2-mercaptoethanol and “complete, EDTA-free” protease inhibitor cocktail tablets (Roche Molecular Biochemicals). .. Lysates were sonicated with three 30-s bursts on a Branson Sonifier 450 (Branson Ultrasonics) and clarified by spinning at 20,000 rpm for 15 min For the first metal affinity purification, 5 ml Ni-NTA resin (QIAGEN) was equilibrated in “binding buffer,” containing 25 mM phosphate, 20 mM Tris-Cl, 10% glycerol, 0.3 M KCl, 5 mM 2-mercaptoethanol, and 0.5% Triton X-100, pH 8.0.

Article Title: Acetate Availability and Utilization Supports the Growth of Mutant Sub-Populations on Aging Bacterial Colonies
Article Snippet: .. The cells were pelleted and lysed by the addition of 400 µl lysis buffer (20 mM HEPES pH 8.0, 9 M urea) containing a protease inhibitor cocktail added according to the manufacturers instructions (Complete, Mini, EDTA-free, Roche Diagnostics Scandinavia AB, Bromma, Sweden) and mixed by pipetting. .. The samples were sonicated with a 16 micron probe for 3×20 seconds, and cooled on ice 1 minute between each sonication.

Article Title: JAGGED Controls Growth Anisotropy and Coordination between Cell Size and Cell Cycle during Plant Organogenesis
Article Snippet: .. Chromatin Immunoprecipitation JAG-GR was activated as described above and ChIP was as described [ ], except for the following modifications: 70–80 inflorescence apices were used per sample; fixation buffer was modified to pH 8.5 and phenylmethylsulfonyl fluoride (PMSF) used at 0.1 mM; after grinding in liquid nitrogen, 300–500 mg tissue powder was resuspended in 700 μl of lysis buffer, modified to contain 0.1 mM PMSF and two tablets of protease inhibitor cocktail complete Mini, EDTA-free (Roche) added per 50 ml of buffer; the supernatant after sonication was precleared for 2 hr with 25 μl Dynabeads Protein A beads (Invitrogen) equilibrated in lysis buffer with 1 mg/ml BSA and 20 μg/ml sonicated salmon sperm, then incubated with 2 μl GR-antibodies (AB3580, Abcam) per 100 μl lysate at 4°C, overnight; 15 μl of equilibrated Dynabeads Protein A beads were added per 100 μl lysate and incubated at 4°C for 4 hr, before proceeding with washes and decrosslinking as described [ ]. ..

Article Title: MicroRNA‐containing extracellular vesicles released from endothelial colony‐forming cells modulate angiogenesis during ischaemic retinopathy
Article Snippet: .. Protein extraction EV protein was extracted using radioimmunoprecipitation assay (RIPA) buffer supplemented with cOmplete Mini, EDTA‐free, Protease Inhibitor Cocktail (Roche, Burgess Hill, UK). .. Quantification was performed using a Micro BCA™ Protein Assay Reagent Kit (Thermo Fisher Scientific).

Article Title: Complement factor H protects mice from ischemic acute kidney injury but is not critical for controlling complement activation by glomerular IgM
Article Snippet: .. Plasma samples were diluted 1:10 in PBS and reduced by boiling with 0.15 M dithiothreitol for 10 min. at 100° C. Renal tissue was homogenized in RIPA lysis buffer containing 1% Triton X-100, 0.5% deoxycholic acid, 150 mM NaCl, 20 mM β-glycerophosphate, 20 mM Tris·HCl (pH 8.0), 5 mM EGTA, 3 mM MgCl2 , 0.1% SDS, 1mM DTT, 50μM Na3 VO4 , and one tablet of complete, EDTA-free, protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN). .. The proteins were resolved by electrophoresis with a 10% Tris-HCl gel (Bio-Rad, Hercules, CA), and transferred to a polyvinylidene fluoride membrane.

Article Title: Identifying protein kinase target preferences using mass spectrometry
Article Snippet: .. Samples were combined with desired concentration of the kinase of interest, 2.67 mM ATP (Cell Signaling Technology, Danvers, MA), and EDTA-free 1 × Complete Mini Protease Inhibitor Cocktail (Roche). .. Samples were brought up to a volume of 150 μl in H2 O to achieve a 1 × kinase reaction mix concentration (50 mM Tris·HCl, 10 mM MgCl2 ) and a rat-tissue protein concentration of 6 μg/μl.

Article Title: Characterization of the Helicase Activity and Anti-telomerase Properties of Yeast Pif1p In Vitro
Article Snippet: .. Protease inhibitor cocktail tablets, EDTA free (Roche Applied Science). ..

Article Title: Insights into Herpesvirus Tegument Organization from Structural Analyses of the 970 Central Residues of HSV-1 UL36 Protein
Article Snippet: .. The pellet was resuspended in 50 m m HEPES, pH 7.0, 500 m m NaCl, 10% glycerol, 1 m m DTT supplemented by a tablet of EDTA-free “Complete” protease inhibitor mixture tablet (Roche Diagnostics) and lysed by sonication at 4 °C. .. The supernatant was recovered and centrifuged at 4 °C for 30 min at 48,000 × g in a JA25.50 rotor (Beckman Coulter).

Article Title: Proteomic Analysis of Detergent Resistant Membrane Domains during Early Interaction of Macrophages with Rough and Smooth Brucella melitensis
Article Snippet: .. The protease inhibitor cocktail Set III, EDTA-free was from Roche. .. SuperSignal West Dura Extended Duration Substrate for the development of Western Blots was from PIERCE.

Article Title: Intrabodies binding the proline-rich domains of mutant huntingtin increase its turnover and reduce neurotoxicity
Article Snippet: .. Cells were dislodged by mechanical dissociation and pipetting 48 hours post-transfection, harvested by centrifugation, washed with PBS and lysed by sonication in 500 μl lysis buffer (25 mM Hepes, 50 mM NaCl, 1 mM MgCl2 , 0.5 % triton) containing 1 Complete, Mini, EDTA-free; Protease Inhibitor Cocktail tablet (Roche) per 7 ml buffer. .. The soluble protein fraction was collected by centrifugation for 20 min at 4° C at 20,000x g. The insoluble pellet was sonicated in 150 μl 6 M urea and incubated for 20 min at RT.

Cell Culture:

Article Title: WT1-AS promotes cell apoptosis in hepatocellular carcinoma through down-regulating of WT1
Article Snippet: .. Protein analysis For Western-blots, total proteins were extracted from tissues or cultured cells using RIPA buffer containing protease inhibitors cOmplete, ULTRA, Mini, EDTA-free, EASYpack (Roche, Basel, Switzerland), while the membrane proteins were extracted from tissues by Mem-PER Eukaryotic Membrane Protein Extraction Reagent Kit (Thermo Scientific, Rockford, USA). ..

Article Title: A Disintegrin-Like and Metalloprotease Domain Containing Thrombospondin Type 1 Motif-like 5 (ADAMTSL5) is a novel fibrillin-1-, fibrillin-2-, and heparin-binding member of the ADAMTS superfamily containing a netrin-like module
Article Snippet: Conditioned 293 SFM II medium was collected from HEK293F cells stably expressing myc-His6 tagged ADAMTSL5 and cultured at 37 °C in 8% CO2 . .. The beads were washed thrice with 0.1% Triton X-100 in PBS, and mixed with 200 μL of 5 μg/mL His6 tagged fibrillin-1 polypeptides rFBN1-N or rFBN1-C representing the N- and C-terminal halves of human fibrillin-1, in Tris-buffered saline with 0.1% Triton X-100 containing EDTA-free, complete mini-protease inhibitor cocktail (Roche) at RT for three hours.

Article Title: pVHL-mediated regulation of the anti-angiogenic protein thrombospondin-1 decreases migration of Clear Cell Renal Carcinoma Cell Lines
Article Snippet: Conditioned media preparation For the detection of secreted TSP-1, cells were cultured for 48 hours in DMEM depleted of FBS. .. Afterwards, conditioned media were collected and complete, EDTA-free, protease inhibitors (Roche, Indianapolis, IN) were added.

DNA Sequencing:

Article Title: Effect of MyBP-C Binding to Actin on Contractility in Heart Muscle
Article Snippet: Automated cycle sequencing was performed by the University of Pennsylvania DNA sequencing facility. .. Cells, collected by centrifugation, were lysed in 50 ml of B-PER bacterial protein extraction reagent (Pierce Chemical Co.), with the addition of 0.3 M NaCl, 5 mM 2-mercaptoethanol and “complete, EDTA-free” protease inhibitor cocktail tablets (Roche Molecular Biochemicals).

Protein Concentration:

Article Title: Identifying protein kinase target preferences using mass spectrometry
Article Snippet: Samples were combined with desired concentration of the kinase of interest, 2.67 mM ATP (Cell Signaling Technology, Danvers, MA), and EDTA-free 1 × Complete Mini Protease Inhibitor Cocktail (Roche). .. Samples were brought up to a volume of 150 μl in H2 O to achieve a 1 × kinase reaction mix concentration (50 mM Tris·HCl, 10 mM MgCl2 ) and a rat-tissue protein concentration of 6 μg/μl.

Sequencing:

Article Title: Effect of MyBP-C Binding to Actin on Contractility in Heart Muscle
Article Snippet: The sequencing primer was designed for the promoter area of pQE-80L plasmid. .. Cells, collected by centrifugation, were lysed in 50 ml of B-PER bacterial protein extraction reagent (Pierce Chemical Co.), with the addition of 0.3 M NaCl, 5 mM 2-mercaptoethanol and “complete, EDTA-free” protease inhibitor cocktail tablets (Roche Molecular Biochemicals).

Sonication:

Article Title: Effect of MyBP-C Binding to Actin on Contractility in Heart Muscle
Article Snippet: Cells, collected by centrifugation, were lysed in 50 ml of B-PER bacterial protein extraction reagent (Pierce Chemical Co.), with the addition of 0.3 M NaCl, 5 mM 2-mercaptoethanol and “complete, EDTA-free” protease inhibitor cocktail tablets (Roche Molecular Biochemicals). .. Lysates were sonicated with three 30-s bursts on a Branson Sonifier 450 (Branson Ultrasonics) and clarified by spinning at 20,000 rpm for 15 min For the first metal affinity purification, 5 ml Ni-NTA resin (QIAGEN) was equilibrated in “binding buffer,” containing 25 mM phosphate, 20 mM Tris-Cl, 10% glycerol, 0.3 M KCl, 5 mM 2-mercaptoethanol, and 0.5% Triton X-100, pH 8.0.

Article Title: Acetate Availability and Utilization Supports the Growth of Mutant Sub-Populations on Aging Bacterial Colonies
Article Snippet: The cells were pelleted and lysed by the addition of 400 µl lysis buffer (20 mM HEPES pH 8.0, 9 M urea) containing a protease inhibitor cocktail added according to the manufacturers instructions (Complete, Mini, EDTA-free, Roche Diagnostics Scandinavia AB, Bromma, Sweden) and mixed by pipetting. .. The samples were sonicated with a 16 micron probe for 3×20 seconds, and cooled on ice 1 minute between each sonication.

Article Title: JAGGED Controls Growth Anisotropy and Coordination between Cell Size and Cell Cycle during Plant Organogenesis
Article Snippet: .. Chromatin Immunoprecipitation JAG-GR was activated as described above and ChIP was as described [ ], except for the following modifications: 70–80 inflorescence apices were used per sample; fixation buffer was modified to pH 8.5 and phenylmethylsulfonyl fluoride (PMSF) used at 0.1 mM; after grinding in liquid nitrogen, 300–500 mg tissue powder was resuspended in 700 μl of lysis buffer, modified to contain 0.1 mM PMSF and two tablets of protease inhibitor cocktail complete Mini, EDTA-free (Roche) added per 50 ml of buffer; the supernatant after sonication was precleared for 2 hr with 25 μl Dynabeads Protein A beads (Invitrogen) equilibrated in lysis buffer with 1 mg/ml BSA and 20 μg/ml sonicated salmon sperm, then incubated with 2 μl GR-antibodies (AB3580, Abcam) per 100 μl lysate at 4°C, overnight; 15 μl of equilibrated Dynabeads Protein A beads were added per 100 μl lysate and incubated at 4°C for 4 hr, before proceeding with washes and decrosslinking as described [ ]. ..

Article Title: Insights into Herpesvirus Tegument Organization from Structural Analyses of the 970 Central Residues of HSV-1 UL36 Protein
Article Snippet: .. The pellet was resuspended in 50 m m HEPES, pH 7.0, 500 m m NaCl, 10% glycerol, 1 m m DTT supplemented by a tablet of EDTA-free “Complete” protease inhibitor mixture tablet (Roche Diagnostics) and lysed by sonication at 4 °C. .. The supernatant was recovered and centrifuged at 4 °C for 30 min at 48,000 × g in a JA25.50 rotor (Beckman Coulter).

Article Title: Thermodynamic Protein Destabilization by GFP Tagging: A Case of Interdomain Allostery
Article Snippet: Purification of eGFP was carried out by resuspending the cells in 50 mM Tris buffer (pH 7.5) containing 100 mM NaCl, 10 mM imidazole, 0.1 mM DTT (buffer B), and the cOmplete, EDTA-free, inhibitor cocktail (Roche Applied Science). .. The cells were then disrupted using a French press and sonication and the lysate was clarified by centrifugation.

Article Title: Intrabodies binding the proline-rich domains of mutant huntingtin increase its turnover and reduce neurotoxicity
Article Snippet: .. Cells were dislodged by mechanical dissociation and pipetting 48 hours post-transfection, harvested by centrifugation, washed with PBS and lysed by sonication in 500 μl lysis buffer (25 mM Hepes, 50 mM NaCl, 1 mM MgCl2 , 0.5 % triton) containing 1 Complete, Mini, EDTA-free; Protease Inhibitor Cocktail tablet (Roche) per 7 ml buffer. .. The soluble protein fraction was collected by centrifugation for 20 min at 4° C at 20,000x g. The insoluble pellet was sonicated in 150 μl 6 M urea and incubated for 20 min at RT.

Affinity Purification:

Article Title: Effect of MyBP-C Binding to Actin on Contractility in Heart Muscle
Article Snippet: Cells, collected by centrifugation, were lysed in 50 ml of B-PER bacterial protein extraction reagent (Pierce Chemical Co.), with the addition of 0.3 M NaCl, 5 mM 2-mercaptoethanol and “complete, EDTA-free” protease inhibitor cocktail tablets (Roche Molecular Biochemicals). .. Lysates were sonicated with three 30-s bursts on a Branson Sonifier 450 (Branson Ultrasonics) and clarified by spinning at 20,000 rpm for 15 min For the first metal affinity purification, 5 ml Ni-NTA resin (QIAGEN) was equilibrated in “binding buffer,” containing 25 mM phosphate, 20 mM Tris-Cl, 10% glycerol, 0.3 M KCl, 5 mM 2-mercaptoethanol, and 0.5% Triton X-100, pH 8.0.

Binding Assay:

Article Title: Effect of MyBP-C Binding to Actin on Contractility in Heart Muscle
Article Snippet: Cells, collected by centrifugation, were lysed in 50 ml of B-PER bacterial protein extraction reagent (Pierce Chemical Co.), with the addition of 0.3 M NaCl, 5 mM 2-mercaptoethanol and “complete, EDTA-free” protease inhibitor cocktail tablets (Roche Molecular Biochemicals). .. Lysates were sonicated with three 30-s bursts on a Branson Sonifier 450 (Branson Ultrasonics) and clarified by spinning at 20,000 rpm for 15 min For the first metal affinity purification, 5 ml Ni-NTA resin (QIAGEN) was equilibrated in “binding buffer,” containing 25 mM phosphate, 20 mM Tris-Cl, 10% glycerol, 0.3 M KCl, 5 mM 2-mercaptoethanol, and 0.5% Triton X-100, pH 8.0.

Article Title: Analysis of Histones and Chromatin in Xenopus laevis Egg and Oocyte Extracts
Article Snippet: .. 10× ELB salt solution: 25 mM MgCl2 , 500 mM KCl, 100 mM Hepes-KOH pH 7.7 Dithiothreitol (DTT, 1 M stock in dH2 O, store at −20 °C) Heparin Sepharose CL-6B, GE Healthcare 17-0467-01 Heparin binding buffer: 1× ELB salts, 10% glycerol, 1 mM EDTA, 1 mM DTT Heparin wash buffer: 1× ELB salts, 0.5 M NaCl 10% glycerol, 1 mM EDTA, 1 mM DTT Heparin elution buffer: 1× ELB salts, 2 M NaCl 10% glycerol, 1 mM EDTA, 1 mM DTT Complete protease inhibitors, EDTA-free, Roche 11 873 850 001 PhosSTOP phostphatase inhibitors, Roche 04 906 837 001 Butyric acid, Aldrich B103500 β-glycerophosphate, Calbiochem 35675 Sodium Fluoride, Fisher S299 Phenylmethanesulphonyl fluoride (PMSF, 100 mM stock in isopropanol), Sigma P7626 Econo-Pac chromatography columns, Bio-Rad #732-1010 β-mercaptoethanol (βME), Fisher 100% trichloroacetic acid (TCA), Fisher A322 Oakridge centrifuge tubes, Nalgene #3119-0050 Refrigerated centrifuge (Sorvall SS-34 rotor) Coomassie Brilliant Blue stain: 40% methanol, 10% acetic acid, 0.025% Coomassie R-250 in dH2 O ..

Molecular Weight:

Article Title: Proteomic Analysis of Detergent Resistant Membrane Domains during Early Interaction of Macrophages with Rough and Smooth Brucella melitensis
Article Snippet: The protease inhibitor cocktail Set III, EDTA-free was from Roche. .. Vivaspin 2 Hydrosart Columns, 5 kDa Molecular Weight Cut-Off (MWCO) were from Sartorius Stedim Biotech.

Mutagenesis:

Article Title: Acetate Availability and Utilization Supports the Growth of Mutant Sub-Populations on Aging Bacterial Colonies
Article Snippet: Protein preparation and mass spectrometry measurements Colonies of pure wild-type, pure rpoB P564L mutant or pure ΔrpoS mutant were initiated and aged. .. The cells were pelleted and lysed by the addition of 400 µl lysis buffer (20 mM HEPES pH 8.0, 9 M urea) containing a protease inhibitor cocktail added according to the manufacturers instructions (Complete, Mini, EDTA-free, Roche Diagnostics Scandinavia AB, Bromma, Sweden) and mixed by pipetting.

Labeling:

Article Title: Proteomic Analysis of Detergent Resistant Membrane Domains during Early Interaction of Macrophages with Rough and Smooth Brucella melitensis
Article Snippet: The protease inhibitor cocktail Set III, EDTA-free was from Roche. .. The iTRAQ® 4-plex labeling reagents were from Applied Biosystems.

Purification:

Article Title: Effect of MyBP-C Binding to Actin on Contractility in Heart Muscle
Article Snippet: Cells, collected by centrifugation, were lysed in 50 ml of B-PER bacterial protein extraction reagent (Pierce Chemical Co.), with the addition of 0.3 M NaCl, 5 mM 2-mercaptoethanol and “complete, EDTA-free” protease inhibitor cocktail tablets (Roche Molecular Biochemicals). .. Purified C0C2 fragment was eluted with the same buffer to which imidazole had been added to a final concentration of 220 mM.

Article Title: Characterization of the Helicase Activity and Anti-telomerase Properties of Yeast Pif1p In Vitro
Article Snippet: Protease inhibitor cocktail tablets, EDTA free (Roche Applied Science). .. Talon polyhistidine-Tag purification resin (Clontech) ( see ).

Article Title: Insights into Herpesvirus Tegument Organization from Structural Analyses of the 970 Central Residues of HSV-1 UL36 Protein
Article Snippet: Paragraph title: Cloning, Expression, and Purification of UL36 Fragments ... The pellet was resuspended in 50 m m HEPES, pH 7.0, 500 m m NaCl, 10% glycerol, 1 m m DTT supplemented by a tablet of EDTA-free “Complete” protease inhibitor mixture tablet (Roche Diagnostics) and lysed by sonication at 4 °C.

Article Title: Thermodynamic Protein Destabilization by GFP Tagging: A Case of Interdomain Allostery
Article Snippet: .. Purification of eGFP was carried out by resuspending the cells in 50 mM Tris buffer (pH 7.5) containing 100 mM NaCl, 10 mM imidazole, 0.1 mM DTT (buffer B), and the cOmplete, EDTA-free, inhibitor cocktail (Roche Applied Science). .. The cells were then disrupted using a French press and sonication and the lysate was clarified by centrifugation.

Protein Extraction:

Article Title: Effect of MyBP-C Binding to Actin on Contractility in Heart Muscle
Article Snippet: .. Cells, collected by centrifugation, were lysed in 50 ml of B-PER bacterial protein extraction reagent (Pierce Chemical Co.), with the addition of 0.3 M NaCl, 5 mM 2-mercaptoethanol and “complete, EDTA-free” protease inhibitor cocktail tablets (Roche Molecular Biochemicals). .. Lysates were sonicated with three 30-s bursts on a Branson Sonifier 450 (Branson Ultrasonics) and clarified by spinning at 20,000 rpm for 15 min For the first metal affinity purification, 5 ml Ni-NTA resin (QIAGEN) was equilibrated in “binding buffer,” containing 25 mM phosphate, 20 mM Tris-Cl, 10% glycerol, 0.3 M KCl, 5 mM 2-mercaptoethanol, and 0.5% Triton X-100, pH 8.0.

Article Title: WT1-AS promotes cell apoptosis in hepatocellular carcinoma through down-regulating of WT1
Article Snippet: .. Protein analysis For Western-blots, total proteins were extracted from tissues or cultured cells using RIPA buffer containing protease inhibitors cOmplete, ULTRA, Mini, EDTA-free, EASYpack (Roche, Basel, Switzerland), while the membrane proteins were extracted from tissues by Mem-PER Eukaryotic Membrane Protein Extraction Reagent Kit (Thermo Scientific, Rockford, USA). ..

Article Title: MicroRNA‐containing extracellular vesicles released from endothelial colony‐forming cells modulate angiogenesis during ischaemic retinopathy
Article Snippet: .. Protein extraction EV protein was extracted using radioimmunoprecipitation assay (RIPA) buffer supplemented with cOmplete Mini, EDTA‐free, Protease Inhibitor Cocktail (Roche, Burgess Hill, UK). .. Quantification was performed using a Micro BCA™ Protein Assay Reagent Kit (Thermo Fisher Scientific).

Positron Emission Tomography:

Article Title: Thermodynamic Protein Destabilization by GFP Tagging: A Case of Interdomain Allostery
Article Snippet: eGFP was purified from E. coli Rosetta (DE3) cells harboring the pET-22b plasmid that contains the gene for this protein. .. Purification of eGFP was carried out by resuspending the cells in 50 mM Tris buffer (pH 7.5) containing 100 mM NaCl, 10 mM imidazole, 0.1 mM DTT (buffer B), and the cOmplete, EDTA-free, inhibitor cocktail (Roche Applied Science).

Polyacrylamide Gel Electrophoresis:

Article Title: WT1-AS promotes cell apoptosis in hepatocellular carcinoma through down-regulating of WT1
Article Snippet: Protein analysis For Western-blots, total proteins were extracted from tissues or cultured cells using RIPA buffer containing protease inhibitors cOmplete, ULTRA, Mini, EDTA-free, EASYpack (Roche, Basel, Switzerland), while the membrane proteins were extracted from tissues by Mem-PER Eukaryotic Membrane Protein Extraction Reagent Kit (Thermo Scientific, Rockford, USA). .. Equal amount of proteins (100ug) were separated with 7.5 %/12.5 % sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membrane.

Lysis:

Article Title: Acetate Availability and Utilization Supports the Growth of Mutant Sub-Populations on Aging Bacterial Colonies
Article Snippet: .. The cells were pelleted and lysed by the addition of 400 µl lysis buffer (20 mM HEPES pH 8.0, 9 M urea) containing a protease inhibitor cocktail added according to the manufacturers instructions (Complete, Mini, EDTA-free, Roche Diagnostics Scandinavia AB, Bromma, Sweden) and mixed by pipetting. .. The samples were sonicated with a 16 micron probe for 3×20 seconds, and cooled on ice 1 minute between each sonication.

Article Title: JAGGED Controls Growth Anisotropy and Coordination between Cell Size and Cell Cycle during Plant Organogenesis
Article Snippet: .. Chromatin Immunoprecipitation JAG-GR was activated as described above and ChIP was as described [ ], except for the following modifications: 70–80 inflorescence apices were used per sample; fixation buffer was modified to pH 8.5 and phenylmethylsulfonyl fluoride (PMSF) used at 0.1 mM; after grinding in liquid nitrogen, 300–500 mg tissue powder was resuspended in 700 μl of lysis buffer, modified to contain 0.1 mM PMSF and two tablets of protease inhibitor cocktail complete Mini, EDTA-free (Roche) added per 50 ml of buffer; the supernatant after sonication was precleared for 2 hr with 25 μl Dynabeads Protein A beads (Invitrogen) equilibrated in lysis buffer with 1 mg/ml BSA and 20 μg/ml sonicated salmon sperm, then incubated with 2 μl GR-antibodies (AB3580, Abcam) per 100 μl lysate at 4°C, overnight; 15 μl of equilibrated Dynabeads Protein A beads were added per 100 μl lysate and incubated at 4°C for 4 hr, before proceeding with washes and decrosslinking as described [ ]. ..

Article Title: Complement factor H protects mice from ischemic acute kidney injury but is not critical for controlling complement activation by glomerular IgM
Article Snippet: .. Plasma samples were diluted 1:10 in PBS and reduced by boiling with 0.15 M dithiothreitol for 10 min. at 100° C. Renal tissue was homogenized in RIPA lysis buffer containing 1% Triton X-100, 0.5% deoxycholic acid, 150 mM NaCl, 20 mM β-glycerophosphate, 20 mM Tris·HCl (pH 8.0), 5 mM EGTA, 3 mM MgCl2 , 0.1% SDS, 1mM DTT, 50μM Na3 VO4 , and one tablet of complete, EDTA-free, protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN). .. The proteins were resolved by electrophoresis with a 10% Tris-HCl gel (Bio-Rad, Hercules, CA), and transferred to a polyvinylidene fluoride membrane.

Article Title: Intrabodies binding the proline-rich domains of mutant huntingtin increase its turnover and reduce neurotoxicity
Article Snippet: .. Cells were dislodged by mechanical dissociation and pipetting 48 hours post-transfection, harvested by centrifugation, washed with PBS and lysed by sonication in 500 μl lysis buffer (25 mM Hepes, 50 mM NaCl, 1 mM MgCl2 , 0.5 % triton) containing 1 Complete, Mini, EDTA-free; Protease Inhibitor Cocktail tablet (Roche) per 7 ml buffer. .. The soluble protein fraction was collected by centrifugation for 20 min at 4° C at 20,000x g. The insoluble pellet was sonicated in 150 μl 6 M urea and incubated for 20 min at RT.

Chromatin Immunoprecipitation:

Article Title: JAGGED Controls Growth Anisotropy and Coordination between Cell Size and Cell Cycle during Plant Organogenesis
Article Snippet: .. Chromatin Immunoprecipitation JAG-GR was activated as described above and ChIP was as described [ ], except for the following modifications: 70–80 inflorescence apices were used per sample; fixation buffer was modified to pH 8.5 and phenylmethylsulfonyl fluoride (PMSF) used at 0.1 mM; after grinding in liquid nitrogen, 300–500 mg tissue powder was resuspended in 700 μl of lysis buffer, modified to contain 0.1 mM PMSF and two tablets of protease inhibitor cocktail complete Mini, EDTA-free (Roche) added per 50 ml of buffer; the supernatant after sonication was precleared for 2 hr with 25 μl Dynabeads Protein A beads (Invitrogen) equilibrated in lysis buffer with 1 mg/ml BSA and 20 μg/ml sonicated salmon sperm, then incubated with 2 μl GR-antibodies (AB3580, Abcam) per 100 μl lysate at 4°C, overnight; 15 μl of equilibrated Dynabeads Protein A beads were added per 100 μl lysate and incubated at 4°C for 4 hr, before proceeding with washes and decrosslinking as described [ ]. ..

SDS Page:

Article Title: WT1-AS promotes cell apoptosis in hepatocellular carcinoma through down-regulating of WT1
Article Snippet: Protein analysis For Western-blots, total proteins were extracted from tissues or cultured cells using RIPA buffer containing protease inhibitors cOmplete, ULTRA, Mini, EDTA-free, EASYpack (Roche, Basel, Switzerland), while the membrane proteins were extracted from tissues by Mem-PER Eukaryotic Membrane Protein Extraction Reagent Kit (Thermo Scientific, Rockford, USA). .. Equal amount of proteins (100ug) were separated with 7.5 %/12.5 % sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membrane.

Article Title: A Disintegrin-Like and Metalloprotease Domain Containing Thrombospondin Type 1 Motif-like 5 (ADAMTSL5) is a novel fibrillin-1-, fibrillin-2-, and heparin-binding member of the ADAMTS superfamily containing a netrin-like module
Article Snippet: The beads were washed thrice with 0.1% Triton X-100 in PBS, and mixed with 200 μL of 5 μg/mL His6 tagged fibrillin-1 polypeptides rFBN1-N or rFBN1-C representing the N- and C-terminal halves of human fibrillin-1, in Tris-buffered saline with 0.1% Triton X-100 containing EDTA-free, complete mini-protease inhibitor cocktail (Roche) at RT for three hours. .. The samples were analyzed by 7.5% SDS-PAGE, and western blotting using monoclonal anti-his antibody (R & D) to detect both ADAMTSL5 and fibrillin-1.

Plasmid Preparation:

Article Title: Effect of MyBP-C Binding to Actin on Contractility in Heart Muscle
Article Snippet: The sequencing primer was designed for the promoter area of pQE-80L plasmid. .. Cells, collected by centrifugation, were lysed in 50 ml of B-PER bacterial protein extraction reagent (Pierce Chemical Co.), with the addition of 0.3 M NaCl, 5 mM 2-mercaptoethanol and “complete, EDTA-free” protease inhibitor cocktail tablets (Roche Molecular Biochemicals).

Article Title: Insights into Herpesvirus Tegument Organization from Structural Analyses of the 970 Central Residues of HSV-1 UL36 Protein
Article Snippet: Amino acids 1600–1733 and 760–1733 of strain 17 HSV-1 UL36 (accession number ) were cloned between the BamHI and EcoRI restriction sites of the pGEX-6P1 (GE-Healthcare) expression vector, resulting in fusions of these fragments with an N-terminal cleavable glutathione S -transferase. .. The pellet was resuspended in 50 m m HEPES, pH 7.0, 500 m m NaCl, 10% glycerol, 1 m m DTT supplemented by a tablet of EDTA-free “Complete” protease inhibitor mixture tablet (Roche Diagnostics) and lysed by sonication at 4 °C.

Article Title: Thermodynamic Protein Destabilization by GFP Tagging: A Case of Interdomain Allostery
Article Snippet: eGFP was purified from E. coli Rosetta (DE3) cells harboring the pET-22b plasmid that contains the gene for this protein. .. Purification of eGFP was carried out by resuspending the cells in 50 mM Tris buffer (pH 7.5) containing 100 mM NaCl, 10 mM imidazole, 0.1 mM DTT (buffer B), and the cOmplete, EDTA-free, inhibitor cocktail (Roche Applied Science).

Article Title: Intrabodies binding the proline-rich domains of mutant huntingtin increase its turnover and reduce neurotoxicity
Article Snippet: Each dish received 4 μg of PQ103 or PQ25 DNA in pcDNA3.1 vector and intrabody DNA in AAV vector at the optimal ratio for each intrabody (4 μg VL 12.3, 16 μg MW7, 8 μg Happ1 and Happ3). .. Cells were dislodged by mechanical dissociation and pipetting 48 hours post-transfection, harvested by centrifugation, washed with PBS and lysed by sonication in 500 μl lysis buffer (25 mM Hepes, 50 mM NaCl, 1 mM MgCl2 , 0.5 % triton) containing 1 Complete, Mini, EDTA-free; Protease Inhibitor Cocktail tablet (Roche) per 7 ml buffer.

Software:

Article Title: Acetate Availability and Utilization Supports the Growth of Mutant Sub-Populations on Aging Bacterial Colonies
Article Snippet: The cells were pelleted and lysed by the addition of 400 µl lysis buffer (20 mM HEPES pH 8.0, 9 M urea) containing a protease inhibitor cocktail added according to the manufacturers instructions (Complete, Mini, EDTA-free, Roche Diagnostics Scandinavia AB, Bromma, Sweden) and mixed by pipetting. .. An LTQ-Orbitrap Velos Pro ETD mass spectrometer (Thermo Fisher Scientific) was used for the mass spectrometry measurements and the PinPoint 1.3 software was used for the quantitation analysis .

Negative Control:

Article Title: Intrabodies binding the proline-rich domains of mutant huntingtin increase its turnover and reduce neurotoxicity
Article Snippet: A non-transfected dish was used as a negative control. .. Cells were dislodged by mechanical dissociation and pipetting 48 hours post-transfection, harvested by centrifugation, washed with PBS and lysed by sonication in 500 μl lysis buffer (25 mM Hepes, 50 mM NaCl, 1 mM MgCl2 , 0.5 % triton) containing 1 Complete, Mini, EDTA-free; Protease Inhibitor Cocktail tablet (Roche) per 7 ml buffer.

Recombinant:

Article Title: Effect of MyBP-C Binding to Actin on Contractility in Heart Muscle
Article Snippet: Paragraph title: Expression of Recombinant C0C2 ... Cells, collected by centrifugation, were lysed in 50 ml of B-PER bacterial protein extraction reagent (Pierce Chemical Co.), with the addition of 0.3 M NaCl, 5 mM 2-mercaptoethanol and “complete, EDTA-free” protease inhibitor cocktail tablets (Roche Molecular Biochemicals).

Radio Immunoprecipitation:

Article Title: MicroRNA‐containing extracellular vesicles released from endothelial colony‐forming cells modulate angiogenesis during ischaemic retinopathy
Article Snippet: .. Protein extraction EV protein was extracted using radioimmunoprecipitation assay (RIPA) buffer supplemented with cOmplete Mini, EDTA‐free, Protease Inhibitor Cocktail (Roche, Burgess Hill, UK). .. Quantification was performed using a Micro BCA™ Protein Assay Reagent Kit (Thermo Fisher Scientific).

Selection:

Article Title: Effect of MyBP-C Binding to Actin on Contractility in Heart Muscle
Article Snippet: After selection of the appropriate clone, the cells were grown in 1 liter of medium to an optical density of 0.7–0.8 at 600 nm, and expression of recombinant His-tagged C0C2 was induced with 1 mM IPTG (Isopropyl-D-thiogalactopyranoside; Roche Molecular Biochemicals) for 4 h at 37°C or overnight at 30°C and then centrifuged at 4°C for 30 min at 3,000 rpm. .. Cells, collected by centrifugation, were lysed in 50 ml of B-PER bacterial protein extraction reagent (Pierce Chemical Co.), with the addition of 0.3 M NaCl, 5 mM 2-mercaptoethanol and “complete, EDTA-free” protease inhibitor cocktail tablets (Roche Molecular Biochemicals).

Incubation:

Article Title: JAGGED Controls Growth Anisotropy and Coordination between Cell Size and Cell Cycle during Plant Organogenesis
Article Snippet: .. Chromatin Immunoprecipitation JAG-GR was activated as described above and ChIP was as described [ ], except for the following modifications: 70–80 inflorescence apices were used per sample; fixation buffer was modified to pH 8.5 and phenylmethylsulfonyl fluoride (PMSF) used at 0.1 mM; after grinding in liquid nitrogen, 300–500 mg tissue powder was resuspended in 700 μl of lysis buffer, modified to contain 0.1 mM PMSF and two tablets of protease inhibitor cocktail complete Mini, EDTA-free (Roche) added per 50 ml of buffer; the supernatant after sonication was precleared for 2 hr with 25 μl Dynabeads Protein A beads (Invitrogen) equilibrated in lysis buffer with 1 mg/ml BSA and 20 μg/ml sonicated salmon sperm, then incubated with 2 μl GR-antibodies (AB3580, Abcam) per 100 μl lysate at 4°C, overnight; 15 μl of equilibrated Dynabeads Protein A beads were added per 100 μl lysate and incubated at 4°C for 4 hr, before proceeding with washes and decrosslinking as described [ ]. ..

Article Title: A Disintegrin-Like and Metalloprotease Domain Containing Thrombospondin Type 1 Motif-like 5 (ADAMTSL5) is a novel fibrillin-1-, fibrillin-2-, and heparin-binding member of the ADAMTS superfamily containing a netrin-like module
Article Snippet: After adjustment to pH 7.2 and addition of phenylmethanesulfonylfluoride (1mM) and Triton X-100 (0.1% v/v), the medium was incubated overnight with anti-myc conjugated agarose beads at 4 °C. .. The beads were washed thrice with 0.1% Triton X-100 in PBS, and mixed with 200 μL of 5 μg/mL His6 tagged fibrillin-1 polypeptides rFBN1-N or rFBN1-C representing the N- and C-terminal halves of human fibrillin-1, in Tris-buffered saline with 0.1% Triton X-100 containing EDTA-free, complete mini-protease inhibitor cocktail (Roche) at RT for three hours.

Article Title: Identifying protein kinase target preferences using mass spectrometry
Article Snippet: Paragraph title: Kinase incubation. ... Samples were combined with desired concentration of the kinase of interest, 2.67 mM ATP (Cell Signaling Technology, Danvers, MA), and EDTA-free 1 × Complete Mini Protease Inhibitor Cocktail (Roche).

Article Title: Intrabodies binding the proline-rich domains of mutant huntingtin increase its turnover and reduce neurotoxicity
Article Snippet: Cells were dislodged by mechanical dissociation and pipetting 48 hours post-transfection, harvested by centrifugation, washed with PBS and lysed by sonication in 500 μl lysis buffer (25 mM Hepes, 50 mM NaCl, 1 mM MgCl2 , 0.5 % triton) containing 1 Complete, Mini, EDTA-free; Protease Inhibitor Cocktail tablet (Roche) per 7 ml buffer. .. The soluble protein fraction was collected by centrifugation for 20 min at 4° C at 20,000x g. The insoluble pellet was sonicated in 150 μl 6 M urea and incubated for 20 min at RT.

Produced:

Article Title: Insights into Herpesvirus Tegument Organization from Structural Analyses of the 970 Central Residues of HSV-1 UL36 Protein
Article Snippet: Both fragments were produced in Escherichia coli BL21 (DE3) cells (Stratagene). .. The pellet was resuspended in 50 m m HEPES, pH 7.0, 500 m m NaCl, 10% glycerol, 1 m m DTT supplemented by a tablet of EDTA-free “Complete” protease inhibitor mixture tablet (Roche Diagnostics) and lysed by sonication at 4 °C.

Concentration Assay:

Article Title: Effect of MyBP-C Binding to Actin on Contractility in Heart Muscle
Article Snippet: Cells, collected by centrifugation, were lysed in 50 ml of B-PER bacterial protein extraction reagent (Pierce Chemical Co.), with the addition of 0.3 M NaCl, 5 mM 2-mercaptoethanol and “complete, EDTA-free” protease inhibitor cocktail tablets (Roche Molecular Biochemicals). .. Purified C0C2 fragment was eluted with the same buffer to which imidazole had been added to a final concentration of 220 mM.

Article Title: Identifying protein kinase target preferences using mass spectrometry
Article Snippet: .. Samples were combined with desired concentration of the kinase of interest, 2.67 mM ATP (Cell Signaling Technology, Danvers, MA), and EDTA-free 1 × Complete Mini Protease Inhibitor Cocktail (Roche). .. Samples were brought up to a volume of 150 μl in H2 O to achieve a 1 × kinase reaction mix concentration (50 mM Tris·HCl, 10 mM MgCl2 ) and a rat-tissue protein concentration of 6 μg/μl.

Stripping:

Article Title: Intrabodies binding the proline-rich domains of mutant huntingtin increase its turnover and reduce neurotoxicity
Article Snippet: Cells were dislodged by mechanical dissociation and pipetting 48 hours post-transfection, harvested by centrifugation, washed with PBS and lysed by sonication in 500 μl lysis buffer (25 mM Hepes, 50 mM NaCl, 1 mM MgCl2 , 0.5 % triton) containing 1 Complete, Mini, EDTA-free; Protease Inhibitor Cocktail tablet (Roche) per 7 ml buffer. .. For a loading control, membranes were stripped using Restore western blot stripping buffer (Pierce) and re-probed with mouse anti-β-tubulin (1:1000 Sigma) as primary and HRP-conjugated, goat anti-mouse (1:10,000 Santa Cruz Biotechnology) as secondary antibody.

Amplex Red Cholesterol Assay:

Article Title: Proteomic Analysis of Detergent Resistant Membrane Domains during Early Interaction of Macrophages with Rough and Smooth Brucella melitensis
Article Snippet: The protease inhibitor cocktail Set III, EDTA-free was from Roche. .. The protease inhibitor cocktail Set III, EDTA-free was from Roche.

Staining:

Article Title: Analysis of Histones and Chromatin in Xenopus laevis Egg and Oocyte Extracts
Article Snippet: .. 10× ELB salt solution: 25 mM MgCl2 , 500 mM KCl, 100 mM Hepes-KOH pH 7.7 Dithiothreitol (DTT, 1 M stock in dH2 O, store at −20 °C) Heparin Sepharose CL-6B, GE Healthcare 17-0467-01 Heparin binding buffer: 1× ELB salts, 10% glycerol, 1 mM EDTA, 1 mM DTT Heparin wash buffer: 1× ELB salts, 0.5 M NaCl 10% glycerol, 1 mM EDTA, 1 mM DTT Heparin elution buffer: 1× ELB salts, 2 M NaCl 10% glycerol, 1 mM EDTA, 1 mM DTT Complete protease inhibitors, EDTA-free, Roche 11 873 850 001 PhosSTOP phostphatase inhibitors, Roche 04 906 837 001 Butyric acid, Aldrich B103500 β-glycerophosphate, Calbiochem 35675 Sodium Fluoride, Fisher S299 Phenylmethanesulphonyl fluoride (PMSF, 100 mM stock in isopropanol), Sigma P7626 Econo-Pac chromatography columns, Bio-Rad #732-1010 β-mercaptoethanol (βME), Fisher 100% trichloroacetic acid (TCA), Fisher A322 Oakridge centrifuge tubes, Nalgene #3119-0050 Refrigerated centrifuge (Sorvall SS-34 rotor) Coomassie Brilliant Blue stain: 40% methanol, 10% acetic acid, 0.025% Coomassie R-250 in dH2 O ..

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  • 92
    Roche edta free proteinase inhibitor cocktail
    Nephrin, cadherins, p120 catenin, and CD2AP co-precipitate in MDCK-nephrin cells. A and B: Cadherins ( lanes 1 and 2 in A ) and p120 catenin ( lane 1 in B ) co-immunoprecipitate with nephrin. No cadherins or p120 catenin can be detected in precipitates obtained with preimmune serum ( lane 3 in A , lane 2 in B ). MDCK-nephrin cell lysate (100 μg, lane 4 in A ; 10 μg, lane 3 in B ) is included as control. MDCK-nephrin cells were lysed in 0.5% Nonidet P-40, 10 <t>mmol/L</t> Tris-HCl, pH 7.6, 150 mmol/L NaCl, 1 mmol/L <t>EDTA</t> with proteinase inhibitors. Immunoprecipitations were performed on ∼2 mg cell lysate with anti-nephrin 029, 1054 ( lanes 1 and 2 in A ), and 1109 ( lane 1 in B ), or preimmune IgG as described in Materials and Methods. Precipitated proteins were separated by 7.5% SDS-PAGE and immunoblotted with anti-pan-cadherin, anti-p120 catenin, or anti-nephrin 043 IgG. C: P-cadherin co-immunoprecipitates with nephrin ( lanes 1 and 2 , middle ) and CD2AP ( lane 3 , middle ). Nephrin can also be immunoprecipitated with anti-CD2AP ( lane 4 , top ). No P-cadherin or nephrin can be detected in precipitates obtained with preimmune serum ( lane 5 ). MDCK-nephrin cell lysate (5 μg in top and middle panels; 25 μg in bottom panel, lane 6 ) is included as control. Immunoprecipitations were performed as described above with the indicated antibodies to nephrin and CD2AP, and the precipitated proteins were analyzed by immunoblotting with anti-nephrin 043, anti-P-cadherin, or anti-CD2AP 1774.
    Edta Free Proteinase Inhibitor Cocktail, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/edta free proteinase inhibitor cocktail/product/Roche
    Average 92 stars, based on 19 article reviews
    Price from $9.99 to $1999.99
    edta free proteinase inhibitor cocktail - by Bioz Stars, 2020-04
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    Roche edta free protease inhibitor cocktail tablets
    Identification of the antigen recognized by mAb B4. A , Lysates prepared from isolated IEC were separated using a sequential three gel separation protocol as described in Materials and Methods . The presence of the B4 antigen was confirmed by Western blot analysis ( right ). Two gel samples ( gray color boxes ) were analyzed by MS. B , The MASCOT search results for the protein isolated from first sample was identified as mouse annexin IV based on the highest score number of 785; 78 matched peptides covering 53% of the annexin IV sequence were identified ( underlying bold letters ). C , Lysates of untransformed F-293 cells ( lane 1 ) and F-293 cells transformed with the pSecTag2/Hygro B expression vector carrying annexin IV insert (F-293-A4) ( lane 3 ) together with culture supernatants from transformed cells ( lane 2 ) were probed by Western blot analysis with mAb B4. D , Flow cytometric analysis of F-293 cells expressing recombinant annexin IV. F-293-A4 cells were probed in flow cytometry with mAb B4. E , Supernatant ( lane1 ) and lysate ( lane 2 ) from F-293-A4 cells expressing recombinant annexin IV that had been incubated in <t>PBS</t> alone, or supernatant ( lane 3 ) and lysate ( lane 4 ) from the same cells incubated with 0.5 M <t>EDTA,</t> were probed by Western blot analysis with mAb B4. Recombinant annexin IV was not released from F-293-A4 cells in the absence of free Ca 2+ .
    Edta Free Protease Inhibitor Cocktail Tablets, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 243 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/edta free protease inhibitor cocktail tablets/product/Roche
    Average 99 stars, based on 243 article reviews
    Price from $9.99 to $1999.99
    edta free protease inhibitor cocktail tablets - by Bioz Stars, 2020-04
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    Roche edta free complete protease inhibitor cocktail
    Polysomal phenotype of siRNA-mediated gene knock down of GYS1. (A) Quantitation of siRNA-mediated gene depletion of GYS1 and pGYS1. Extracts from HeLa cells, transfected with 50 nM GYS1 or control siRNAs, were separated by <t>SDS-polyacrylamide</t> gel electrophoresis and pGYS1 and GYS1 abundances examined by Western blot analysis. Actin was used as an internal control. (B) Polysomal distribution in control siRNA- and GYS1 siRNA-treated extracts. Cell lysates, treated as described in (2A) were separated by ultracentrifugation in 10–60% sucrose gradients. Overlays of the 254nm absorbance profiles are shown. (C) Polysome distribution in HeLa cells transfected with empty 3xFLAG vector or 3xFLAG-GYS1 DNA. Overlay of the 254nm absorbance profiles following separation of cells lysates in 10–60% sucrose gradients are shown. (D, E) Quantitation of ribosomal subunits in control siRNA- and GYS1 siRNA-treated extracts. Ribosomal subunits were released from polysomes and dissociated into 40S and 60S subunits by either treatment with 2 mM puromycin (D) or 15 mM <t>EDTA</t> (E). Cell lysates were separated in 10–60% sucrose gradient and absorbance was measured at 254 nm.
    Edta Free Complete Protease Inhibitor Cocktail, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 347 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/edta free complete protease inhibitor cocktail/product/Roche
    Average 99 stars, based on 347 article reviews
    Price from $9.99 to $1999.99
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    Image Search Results


    Nephrin, cadherins, p120 catenin, and CD2AP co-precipitate in MDCK-nephrin cells. A and B: Cadherins ( lanes 1 and 2 in A ) and p120 catenin ( lane 1 in B ) co-immunoprecipitate with nephrin. No cadherins or p120 catenin can be detected in precipitates obtained with preimmune serum ( lane 3 in A , lane 2 in B ). MDCK-nephrin cell lysate (100 μg, lane 4 in A ; 10 μg, lane 3 in B ) is included as control. MDCK-nephrin cells were lysed in 0.5% Nonidet P-40, 10 mmol/L Tris-HCl, pH 7.6, 150 mmol/L NaCl, 1 mmol/L EDTA with proteinase inhibitors. Immunoprecipitations were performed on ∼2 mg cell lysate with anti-nephrin 029, 1054 ( lanes 1 and 2 in A ), and 1109 ( lane 1 in B ), or preimmune IgG as described in Materials and Methods. Precipitated proteins were separated by 7.5% SDS-PAGE and immunoblotted with anti-pan-cadherin, anti-p120 catenin, or anti-nephrin 043 IgG. C: P-cadherin co-immunoprecipitates with nephrin ( lanes 1 and 2 , middle ) and CD2AP ( lane 3 , middle ). Nephrin can also be immunoprecipitated with anti-CD2AP ( lane 4 , top ). No P-cadherin or nephrin can be detected in precipitates obtained with preimmune serum ( lane 5 ). MDCK-nephrin cell lysate (5 μg in top and middle panels; 25 μg in bottom panel, lane 6 ) is included as control. Immunoprecipitations were performed as described above with the indicated antibodies to nephrin and CD2AP, and the precipitated proteins were analyzed by immunoblotting with anti-nephrin 043, anti-P-cadherin, or anti-CD2AP 1774.

    Journal: The American Journal of Pathology

    Article Title: Nephrin Forms a Complex with Adherens Junction Proteins and CASK in Podocytes and in Madin-Darby Canine Kidney Cells Expressing Nephrin

    doi:

    Figure Lengend Snippet: Nephrin, cadherins, p120 catenin, and CD2AP co-precipitate in MDCK-nephrin cells. A and B: Cadherins ( lanes 1 and 2 in A ) and p120 catenin ( lane 1 in B ) co-immunoprecipitate with nephrin. No cadherins or p120 catenin can be detected in precipitates obtained with preimmune serum ( lane 3 in A , lane 2 in B ). MDCK-nephrin cell lysate (100 μg, lane 4 in A ; 10 μg, lane 3 in B ) is included as control. MDCK-nephrin cells were lysed in 0.5% Nonidet P-40, 10 mmol/L Tris-HCl, pH 7.6, 150 mmol/L NaCl, 1 mmol/L EDTA with proteinase inhibitors. Immunoprecipitations were performed on ∼2 mg cell lysate with anti-nephrin 029, 1054 ( lanes 1 and 2 in A ), and 1109 ( lane 1 in B ), or preimmune IgG as described in Materials and Methods. Precipitated proteins were separated by 7.5% SDS-PAGE and immunoblotted with anti-pan-cadherin, anti-p120 catenin, or anti-nephrin 043 IgG. C: P-cadherin co-immunoprecipitates with nephrin ( lanes 1 and 2 , middle ) and CD2AP ( lane 3 , middle ). Nephrin can also be immunoprecipitated with anti-CD2AP ( lane 4 , top ). No P-cadherin or nephrin can be detected in precipitates obtained with preimmune serum ( lane 5 ). MDCK-nephrin cell lysate (5 μg in top and middle panels; 25 μg in bottom panel, lane 6 ) is included as control. Immunoprecipitations were performed as described above with the indicated antibodies to nephrin and CD2AP, and the precipitated proteins were analyzed by immunoblotting with anti-nephrin 043, anti-P-cadherin, or anti-CD2AP 1774.

    Article Snippet: MDCK-nephrin or MDCK-mock cells on 100-mm dishes were washed twice with ice-cold phosphate-buffered saline (PBS) and scraped from the dish with a cell lifter into 1 ml of ice-cold lysis buffer [0.5% Nonidet P-40, 10 mmol/L Tris-HCl, pH 7.6, 150 mmol/L NaCl, 1 mmol/L ethylenediaminetetraacetic acid (EDTA) with or without 0.25% Triton X-100 (TX-100)] supplemented with 1× Complete, EDTA-free proteinase inhibitor cocktail (Roche, Mannheim, Germany), 50 mmol/L sodium fluoride (to inhibit Ser/Thr phosphatases), and 1 mmol/L sodium vanadate (to inhibit tyrosine phosphatases).

    Techniques: SDS Page, Immunoprecipitation

    Identification of the antigen recognized by mAb B4. A , Lysates prepared from isolated IEC were separated using a sequential three gel separation protocol as described in Materials and Methods . The presence of the B4 antigen was confirmed by Western blot analysis ( right ). Two gel samples ( gray color boxes ) were analyzed by MS. B , The MASCOT search results for the protein isolated from first sample was identified as mouse annexin IV based on the highest score number of 785; 78 matched peptides covering 53% of the annexin IV sequence were identified ( underlying bold letters ). C , Lysates of untransformed F-293 cells ( lane 1 ) and F-293 cells transformed with the pSecTag2/Hygro B expression vector carrying annexin IV insert (F-293-A4) ( lane 3 ) together with culture supernatants from transformed cells ( lane 2 ) were probed by Western blot analysis with mAb B4. D , Flow cytometric analysis of F-293 cells expressing recombinant annexin IV. F-293-A4 cells were probed in flow cytometry with mAb B4. E , Supernatant ( lane1 ) and lysate ( lane 2 ) from F-293-A4 cells expressing recombinant annexin IV that had been incubated in PBS alone, or supernatant ( lane 3 ) and lysate ( lane 4 ) from the same cells incubated with 0.5 M EDTA, were probed by Western blot analysis with mAb B4. Recombinant annexin IV was not released from F-293-A4 cells in the absence of free Ca 2+ .

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Pathogenic Natural Antibodies Recognizing Annexin IV Are Required to Develop Intestinal Ischemia-Reperfusion Injury

    doi: 10.4049/jimmunol.0803980

    Figure Lengend Snippet: Identification of the antigen recognized by mAb B4. A , Lysates prepared from isolated IEC were separated using a sequential three gel separation protocol as described in Materials and Methods . The presence of the B4 antigen was confirmed by Western blot analysis ( right ). Two gel samples ( gray color boxes ) were analyzed by MS. B , The MASCOT search results for the protein isolated from first sample was identified as mouse annexin IV based on the highest score number of 785; 78 matched peptides covering 53% of the annexin IV sequence were identified ( underlying bold letters ). C , Lysates of untransformed F-293 cells ( lane 1 ) and F-293 cells transformed with the pSecTag2/Hygro B expression vector carrying annexin IV insert (F-293-A4) ( lane 3 ) together with culture supernatants from transformed cells ( lane 2 ) were probed by Western blot analysis with mAb B4. D , Flow cytometric analysis of F-293 cells expressing recombinant annexin IV. F-293-A4 cells were probed in flow cytometry with mAb B4. E , Supernatant ( lane1 ) and lysate ( lane 2 ) from F-293-A4 cells expressing recombinant annexin IV that had been incubated in PBS alone, or supernatant ( lane 3 ) and lysate ( lane 4 ) from the same cells incubated with 0.5 M EDTA, were probed by Western blot analysis with mAb B4. Recombinant annexin IV was not released from F-293-A4 cells in the absence of free Ca 2+ .

    Article Snippet: After harvesting the cells, they were resuspended in PBS with Complete, EDTA-Free Protease Inhibitor Cocktail Tablets (Roche Molecular Biochemicals).

    Techniques: Isolation, Western Blot, Mass Spectrometry, Sequencing, Transformation Assay, Expressing, Plasmid Preparation, Flow Cytometry, Recombinant, Cytometry, Incubation

    Polysomal phenotype of siRNA-mediated gene knock down of GYS1. (A) Quantitation of siRNA-mediated gene depletion of GYS1 and pGYS1. Extracts from HeLa cells, transfected with 50 nM GYS1 or control siRNAs, were separated by SDS-polyacrylamide gel electrophoresis and pGYS1 and GYS1 abundances examined by Western blot analysis. Actin was used as an internal control. (B) Polysomal distribution in control siRNA- and GYS1 siRNA-treated extracts. Cell lysates, treated as described in (2A) were separated by ultracentrifugation in 10–60% sucrose gradients. Overlays of the 254nm absorbance profiles are shown. (C) Polysome distribution in HeLa cells transfected with empty 3xFLAG vector or 3xFLAG-GYS1 DNA. Overlay of the 254nm absorbance profiles following separation of cells lysates in 10–60% sucrose gradients are shown. (D, E) Quantitation of ribosomal subunits in control siRNA- and GYS1 siRNA-treated extracts. Ribosomal subunits were released from polysomes and dissociated into 40S and 60S subunits by either treatment with 2 mM puromycin (D) or 15 mM EDTA (E). Cell lysates were separated in 10–60% sucrose gradient and absorbance was measured at 254 nm.

    Journal: Journal of molecular biology

    Article Title: Proteomic Analysis of Ribosomes: Translational Control of mRNA populations by Glycogen Synthase GYS1

    doi: 10.1016/j.jmb.2011.04.064

    Figure Lengend Snippet: Polysomal phenotype of siRNA-mediated gene knock down of GYS1. (A) Quantitation of siRNA-mediated gene depletion of GYS1 and pGYS1. Extracts from HeLa cells, transfected with 50 nM GYS1 or control siRNAs, were separated by SDS-polyacrylamide gel electrophoresis and pGYS1 and GYS1 abundances examined by Western blot analysis. Actin was used as an internal control. (B) Polysomal distribution in control siRNA- and GYS1 siRNA-treated extracts. Cell lysates, treated as described in (2A) were separated by ultracentrifugation in 10–60% sucrose gradients. Overlays of the 254nm absorbance profiles are shown. (C) Polysome distribution in HeLa cells transfected with empty 3xFLAG vector or 3xFLAG-GYS1 DNA. Overlay of the 254nm absorbance profiles following separation of cells lysates in 10–60% sucrose gradients are shown. (D, E) Quantitation of ribosomal subunits in control siRNA- and GYS1 siRNA-treated extracts. Ribosomal subunits were released from polysomes and dissociated into 40S and 60S subunits by either treatment with 2 mM puromycin (D) or 15 mM EDTA (E). Cell lysates were separated in 10–60% sucrose gradient and absorbance was measured at 254 nm.

    Article Snippet: For preparation of total cell lysates, HeLa cells were washed and harvested in cold PBS, followed by lysis in RIPA buffer (150 mM NaCl, 1mM EDTA, 100 mM Tris-HCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) containing EDTA-free complete protease inhibitor cocktail (Roche).

    Techniques: Quantitation Assay, Transfection, Polyacrylamide Gel Electrophoresis, Western Blot, Plasmid Preparation

    Characterization of recombinant B. subtilis PhoD. A , phosphate released from different substrates by PhoD. The assays contained 1 μg/ml PhoD and 1 m m of the test substrate in 100 m m Tris, pH 8.0, 2 m m CaCl 2 . Gly3P is glycerol 3-phosphate; Phoac is phosphonoacetic acid; Glu6P is glucose 6-phosphate; imidodiP is imidodiphosphate. Substrates are grouped according to whether the target bond is between phosphorus and oxygen (O–P), phosphorus and carbon (C–P), or phosphorus and nitrogen (N–P). Error bars indicate the S.D. of three independent experiments. B , visible absorption spectrum of 2.3 mg/ml PhoD in 20 m m Hepes NaOH, pH 7.4, 200 m m NaCl, 1 m m CaCl 2 in the absence ( black ) or presence (gray) of 10 m m Na 2 EGTA. C , EPR spectra of PhoD highlighting the g = 4.3 peak. Spectra are shown for native PhoD, PhoD with 5 m m sodium dithionite, PhoD with 2 m m hydrogen peroxide, and PhoD with 50 m m EDTA. The inset shows a magnified part of the spectrum around g = 9.6. The PhoD concentration was 10 mg/ml in 20 m m Hepes, pH 7.5, 400 m m NaCl, 2 m m calcium acetate with 20% (v/v) glycerol added. A control experiment showed that the glycerol added to the samples did not change the spectral line shape (data not shown). EPR operating conditions: microwave power, 2 milliwatts; microwave frequency, 9.425 GHz; modulation, 5.0 G at 100 kHz; temperature, 10 K. Spectra are buffer-subtracted, with negative controls taken for sodium dithionite, hydrogen peroxide, and EDTA solutions.

    Journal: The Journal of Biological Chemistry

    Article Title: Crystal Structure of the Bacillus subtilis Phosphodiesterase PhoD Reveals an Iron and Calcium-containing Active Site ♦

    doi: 10.1074/jbc.M114.604892

    Figure Lengend Snippet: Characterization of recombinant B. subtilis PhoD. A , phosphate released from different substrates by PhoD. The assays contained 1 μg/ml PhoD and 1 m m of the test substrate in 100 m m Tris, pH 8.0, 2 m m CaCl 2 . Gly3P is glycerol 3-phosphate; Phoac is phosphonoacetic acid; Glu6P is glucose 6-phosphate; imidodiP is imidodiphosphate. Substrates are grouped according to whether the target bond is between phosphorus and oxygen (O–P), phosphorus and carbon (C–P), or phosphorus and nitrogen (N–P). Error bars indicate the S.D. of three independent experiments. B , visible absorption spectrum of 2.3 mg/ml PhoD in 20 m m Hepes NaOH, pH 7.4, 200 m m NaCl, 1 m m CaCl 2 in the absence ( black ) or presence (gray) of 10 m m Na 2 EGTA. C , EPR spectra of PhoD highlighting the g = 4.3 peak. Spectra are shown for native PhoD, PhoD with 5 m m sodium dithionite, PhoD with 2 m m hydrogen peroxide, and PhoD with 50 m m EDTA. The inset shows a magnified part of the spectrum around g = 9.6. The PhoD concentration was 10 mg/ml in 20 m m Hepes, pH 7.5, 400 m m NaCl, 2 m m calcium acetate with 20% (v/v) glycerol added. A control experiment showed that the glycerol added to the samples did not change the spectral line shape (data not shown). EPR operating conditions: microwave power, 2 milliwatts; microwave frequency, 9.425 GHz; modulation, 5.0 G at 100 kHz; temperature, 10 K. Spectra are buffer-subtracted, with negative controls taken for sodium dithionite, hydrogen peroxide, and EDTA solutions.

    Article Snippet: Cells were harvested by centrifugation and resuspended in resuspension buffer (20 mm Hepes NaOH, pH 7.4, 200 mm NaCl, 1 mm CaCl2 ), with EDTA-free complete protease inhibitors (Roche Applied Science) and DNase I (Sigma-Aldrich).

    Techniques: Recombinant, Electron Paramagnetic Resonance, Concentration Assay