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Roche lysis buffer
Lysis Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 1428 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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lysis buffer - by Bioz Stars, 2020-04
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Related Articles

Clone Assay:

Article Title: Identification and functional analysis of NOL7 nuclear and nucleolar localization signals
Article Snippet: Protein Purification 293T cells were transfected with the NOL7-GFP purification construct and positive clones were selected and maintained as described. .. Cells were pelleted by centrifugation and resuspended in lysis buffer (50 mM sodium phosphate, pH 7.4; 300 mM NaCl; 1% Triton X-100; Roche Complete EDTA-free protease inhibitor tablet).

Centrifugation:

Article Title: CAEV Vif Hijacks ElonginB/C, CYPA and Cullin5 to Assemble the E3 Ubiquitin Ligase Complex Stepwise to Degrade oaA3Z2-Z3
Article Snippet: At 48 h post transfection, the HEK293T cells were harvested and washed twice with cold PBS, followed by dissociating in lysis buffer (PBS containing 1% Triton X-100 and complete protease inhibitor cocktail tablets [Roche, Mannheim, Germany]) at 4 °C for 30 min. .. The pre-cleared supernatants were collected by centrifugation at 10,000 g for 30 min at 4 °C and then mixed with anti-HA antibody conjugated agarose beads (cCatalog nNo.

Article Title: Identification and functional analysis of NOL7 nuclear and nucleolar localization signals
Article Snippet: .. Cells were pelleted by centrifugation and resuspended in lysis buffer (50 mM sodium phosphate, pH 7.4; 300 mM NaCl; 1% Triton X-100; Roche Complete EDTA-free protease inhibitor tablet). ..

Article Title: Expression, purification, crystallization and preliminary crystallographic analysis of MxiH, a subunit of the Shigella flexneri type III secretion system needle
Article Snippet: .. After ∼16 h, cells were harvested by centrifugation (15 min, 5000g , 277 K) and pellets were frozen at 193 K. Cell pellets were resuspended in lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl and Complete EDTA-free protease inhibitor cocktail, Roche) and lysed using an Emulsiflex-C5 Homogeniser (Glen Creston, UK). .. The resultant cell suspension was centrifuged (20 min, 20 000g , 277 K) and the soluble fraction was applied to a pre-charged HiTrap HP nickel-affinity column (HiTrap Chelating HP, Amersham Biosciences).

Article Title: Inhibition of HCV Replication by Oxysterol-Binding Protein-Related Protein 4 (ORP4) through Interaction with HCV NS5B and Alteration of Lipid Droplet Formation
Article Snippet: Forty-eight hours post transfection, the cell culture supernatants were collected and cell debris was removed by a brief centrifugation. .. Cells were then washed with ice-cold PBS and lysed in 100 µl lysis buffer (1% Triton X-100 and 1X Roche protease inhibitor cocktail in PBS).

Article Title: A NUMB–EFA6B–ARF6 recycling route controls apically restricted cell protrusions and mesenchymal motility
Article Snippet: .. After 15 min of centrifugation at 1,200 rpm, pellets were suspended in lysis buffer (2× TBS, 0.5 mM EDTA, 10% glycerol, protease inhibitor cocktail [Roche; freshly added], and 1 mM DTT [freshly added]; 25 ml for 1-liter culture) or conserved at −80°C after washing in PBS once. ..

Article Title: Nuclear transport adapts to varying heat stress in a multistep mechanism
Article Snippet: Then, the cells were suspended in 500 µl lysis buffer (0.5% Triton X-100, 20 mM N -ethylmaleimide, and protease inhibitor cocktail [11873580001; Roche] in PBS− ) and rotated for 20 min at 4°C. .. After centrifugation, the supernatants were collected as soluble fractions.

Article Title: A two-hit mechanism causes cerebral cavernous malformations: complete inactivation of CCM1, CCM2 or CCM3 in affected endothelial cells
Article Snippet: After a centrifugation step of 1000 r.p.m. for 2 min (rotor F45-30-11, eppendorf, Hamburg), the post-nuclear supernatants were frozen in liquid nitrogen. .. Cell lysates were prepared with lysis buffer (1% Triton-X-100, 10 mm Tris pH 8, 1 mm EDTA, Roche complete EDTA-free protease inhibitor).

Amplification:

Article Title: A NUMB–EFA6B–ARF6 recycling route controls apically restricted cell protrusions and mesenchymal motility
Article Snippet: The virus was amplified a second time using 200 ml of H5 cells infected with 2.5 ml of the V1 virus, following the same protocol. .. After 15 min of centrifugation at 1,200 rpm, pellets were suspended in lysis buffer (2× TBS, 0.5 mM EDTA, 10% glycerol, protease inhibitor cocktail [Roche; freshly added], and 1 mM DTT [freshly added]; 25 ml for 1-liter culture) or conserved at −80°C after washing in PBS once.

Construct:

Article Title: Identification and functional analysis of NOL7 nuclear and nucleolar localization signals
Article Snippet: Protein Purification 293T cells were transfected with the NOL7-GFP purification construct and positive clones were selected and maintained as described. .. Cells were pelleted by centrifugation and resuspended in lysis buffer (50 mM sodium phosphate, pH 7.4; 300 mM NaCl; 1% Triton X-100; Roche Complete EDTA-free protease inhibitor tablet).

Article Title: Expression, purification, crystallization and preliminary crystallographic analysis of MxiH, a subunit of the Shigella flexneri type III secretion system needle
Article Snippet: This construct includes two additional residues (Leu79 and Glu80) that link the truncated form of MxiH to a C-­terminal His6 tag. .. After ∼16 h, cells were harvested by centrifugation (15 min, 5000g , 277 K) and pellets were frozen at 193 K. Cell pellets were resuspended in lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl and Complete EDTA-free protease inhibitor cocktail, Roche) and lysed using an Emulsiflex-C5 Homogeniser (Glen Creston, UK).

Incubation:

Article Title: Knockdown of IKK1/2 Promotes Differentiation of Mouse Embryonic Stem Cells into Neuroectoderm at the Expense of Mesoderm
Article Snippet: Western Blot Cells were extracted in lysis-buffer (1 % SDS, 5 mM EDTA, complete protease inhibitor cocktail (Roche)) directly in the dish on ice. .. Blots were probed with primary antibodies overnight and incubated with horseradish peroxidase-conjugated secondary antibodies the next day for 1 h. Western blots were carried out using GAPDH (Santa Cruz, sc-32233), pospho IκBα (Ser 32/36) (Cell Signaling, 5A5) and Flag (Sigma Aldrich; F7425) primary antibodies and developed using ECL (enhanced chemiluminescence).

Article Title: Co-Regulation of NF-?B and Inflammasome-Mediated Inflammatory Responses by Myxoma Virus Pyrin Domain-Containing Protein M013
Article Snippet: Forty eight hours post transfection, cells were collected in 500 µl PBS, pelleted and then lysed by suspending in 20 µl lysis buffer (100 mM NaCl, 100 mM Tris, pH 8.0, 0.5% NP-40, containing 25 µl/ml Roche complete protease inhibitor). .. Five µl of anti-GST acceptor beads (PerkinElmer, 6760603C), diluted 50-fold (20 µg/ml final concentration of beads in assay) in 0.1% BSA/PBS assay buffer, and 5 µl of cell extract were mixed to a final volume of 20 µl with 0.1% BSA/PBS assay buffer, and incubated at room temperature for 1.5 hour.

Article Title: A NUMB–EFA6B–ARF6 recycling route controls apically restricted cell protrusions and mesenchymal motility
Article Snippet: After 15 min of centrifugation at 1,200 rpm, pellets were suspended in lysis buffer (2× TBS, 0.5 mM EDTA, 10% glycerol, protease inhibitor cocktail [Roche; freshly added], and 1 mM DTT [freshly added]; 25 ml for 1-liter culture) or conserved at −80°C after washing in PBS once. .. 1 ml of amylose beads (NEB), previously washed three times with lysis buffer, was added to the supernatant, and samples were incubated for 1–2 h at 4°C while rocking.

Article Title: Reptin Regulates DNA Double Strand Breaks Repair in Human Hepatocellular Carcinoma
Article Snippet: Briefly, HuH7 cells were incubated in methionine/cysteine-free medium for 1 h before pulse labeling with 150 μCi/mL EXPRE35 S35 S Protein Labeling Mix (Perkin Elmer, Courtaboeuf, France) for 30 min at 37°C. .. Cells were then scraped in lysis buffer (5 PBS with 1% Triton X-100, Complete x1, Roche) and sonicated for 30 s on ice.

Expressing:

Article Title: CAEV Vif Hijacks ElonginB/C, CYPA and Cullin5 to Assemble the E3 Ubiquitin Ligase Complex Stepwise to Degrade oaA3Z2-Z3
Article Snippet: Immunoprecipitation To determine the interaction between CAEV Vif wild type (WT) or its mutants and cellular partners, HEK293T cells were transfected with HA-tagged expression vectors of WT or mutants of CAEV Vif. .. At 48 h post transfection, the HEK293T cells were harvested and washed twice with cold PBS, followed by dissociating in lysis buffer (PBS containing 1% Triton X-100 and complete protease inhibitor cocktail tablets [Roche, Mannheim, Germany]) at 4 °C for 30 min.

Article Title: Co-Regulation of NF-?B and Inflammasome-Mediated Inflammatory Responses by Myxoma Virus Pyrin Domain-Containing Protein M013
Article Snippet: On the other hand, expression from the pANT7_cGST plasmid is under the control of T7 RNA polymerase promoter and expressed proteins are fused at their C-terminal with a glutathione-S-transferase (GST) tag. .. Forty eight hours post transfection, cells were collected in 500 µl PBS, pelleted and then lysed by suspending in 20 µl lysis buffer (100 mM NaCl, 100 mM Tris, pH 8.0, 0.5% NP-40, containing 25 µl/ml Roche complete protease inhibitor).

Article Title: Expression, purification, crystallization and preliminary crystallographic analysis of MxiH, a subunit of the Shigella flexneri type III secretion system needle
Article Snippet: Paragraph title: Expression and purification ... After ∼16 h, cells were harvested by centrifugation (15 min, 5000g , 277 K) and pellets were frozen at 193 K. Cell pellets were resuspended in lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl and Complete EDTA-free protease inhibitor cocktail, Roche) and lysed using an Emulsiflex-C5 Homogeniser (Glen Creston, UK).

Western Blot:

Article Title: Identification and functional analysis of NOL7 nuclear and nucleolar localization signals
Article Snippet: Cells were pelleted by centrifugation and resuspended in lysis buffer (50 mM sodium phosphate, pH 7.4; 300 mM NaCl; 1% Triton X-100; Roche Complete EDTA-free protease inhibitor tablet). .. Fractions of approximately 300 μl were collected and tested for the presence of NOL7 by SDS-PAGE followed by silver staining and western blot using mouse α-V5 monoclonal antibody (Invitrogen, Carlsbad CA).

Article Title: Knockdown of IKK1/2 Promotes Differentiation of Mouse Embryonic Stem Cells into Neuroectoderm at the Expense of Mesoderm
Article Snippet: .. Western Blot Cells were extracted in lysis-buffer (1 % SDS, 5 mM EDTA, complete protease inhibitor cocktail (Roche)) directly in the dish on ice. .. Equal amounts of protein extracts were separated on a 10 % SDS-PAGE and transferred to nitrocellulose membranes (PALL).

Article Title: A two-hit mechanism causes cerebral cavernous malformations: complete inactivation of CCM1, CCM2 or CCM3 in affected endothelial cells
Article Snippet: Paragraph title: Western blotting ... Cell lysates were prepared with lysis buffer (1% Triton-X-100, 10 mm Tris pH 8, 1 mm EDTA, Roche complete EDTA-free protease inhibitor).

Over Expression:

Article Title: Expression, purification, crystallization and preliminary crystallographic analysis of MxiH, a subunit of the Shigella flexneri type III secretion system needle
Article Snippet: Cells were grown at 310 K until an A 280nm of ∼0.6 was reached, whereupon the solutions were cooled to 293 K and protein overexpression was induced by the addition of 1.0 mM IPTG. .. After ∼16 h, cells were harvested by centrifugation (15 min, 5000g , 277 K) and pellets were frozen at 193 K. Cell pellets were resuspended in lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl and Complete EDTA-free protease inhibitor cocktail, Roche) and lysed using an Emulsiflex-C5 Homogeniser (Glen Creston, UK).

Cholesterol Assay:

Article Title: Inhibition of HCV Replication by Oxysterol-Binding Protein-Related Protein 4 (ORP4) through Interaction with HCV NS5B and Alteration of Lipid Droplet Formation
Article Snippet: Paragraph title: Cholesterol assay ... Cells were then washed with ice-cold PBS and lysed in 100 µl lysis buffer (1% Triton X-100 and 1X Roche protease inhibitor cocktail in PBS).

Transfection:

Article Title: CAEV Vif Hijacks ElonginB/C, CYPA and Cullin5 to Assemble the E3 Ubiquitin Ligase Complex Stepwise to Degrade oaA3Z2-Z3
Article Snippet: .. At 48 h post transfection, the HEK293T cells were harvested and washed twice with cold PBS, followed by dissociating in lysis buffer (PBS containing 1% Triton X-100 and complete protease inhibitor cocktail tablets [Roche, Mannheim, Germany]) at 4 °C for 30 min. .. The pre-cleared supernatants were collected by centrifugation at 10,000 g for 30 min at 4 °C and then mixed with anti-HA antibody conjugated agarose beads (cCatalog nNo.

Article Title: Identification and functional analysis of NOL7 nuclear and nucleolar localization signals
Article Snippet: Protein Purification 293T cells were transfected with the NOL7-GFP purification construct and positive clones were selected and maintained as described. .. Cells were pelleted by centrifugation and resuspended in lysis buffer (50 mM sodium phosphate, pH 7.4; 300 mM NaCl; 1% Triton X-100; Roche Complete EDTA-free protease inhibitor tablet).

Article Title: Co-Regulation of NF-?B and Inflammasome-Mediated Inflammatory Responses by Myxoma Virus Pyrin Domain-Containing Protein M013
Article Snippet: .. Forty eight hours post transfection, cells were collected in 500 µl PBS, pelleted and then lysed by suspending in 20 µl lysis buffer (100 mM NaCl, 100 mM Tris, pH 8.0, 0.5% NP-40, containing 25 µl/ml Roche complete protease inhibitor). ..

Article Title: Inhibition of HCV Replication by Oxysterol-Binding Protein-Related Protein 4 (ORP4) through Interaction with HCV NS5B and Alteration of Lipid Droplet Formation
Article Snippet: Forty-eight hours post transfection, the cell culture supernatants were collected and cell debris was removed by a brief centrifugation. .. Cells were then washed with ice-cold PBS and lysed in 100 µl lysis buffer (1% Triton X-100 and 1X Roche protease inhibitor cocktail in PBS).

Chromatography:

Article Title: Expression, purification, crystallization and preliminary crystallographic analysis of MxiH, a subunit of the Shigella flexneri type III secretion system needle
Article Snippet: After ∼16 h, cells were harvested by centrifugation (15 min, 5000g , 277 K) and pellets were frozen at 193 K. Cell pellets were resuspended in lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl and Complete EDTA-free protease inhibitor cocktail, Roche) and lysed using an Emulsiflex-C5 Homogeniser (Glen Creston, UK). .. Fractions containing MxiHCΔ5 were further purified by gel-filtration chromatography using a HiLoad 16/60 Superdex 75 column (Amersham Biosciences).

Immunoprecipitation:

Article Title: CAEV Vif Hijacks ElonginB/C, CYPA and Cullin5 to Assemble the E3 Ubiquitin Ligase Complex Stepwise to Degrade oaA3Z2-Z3
Article Snippet: Paragraph title: Immunoprecipitation ... At 48 h post transfection, the HEK293T cells were harvested and washed twice with cold PBS, followed by dissociating in lysis buffer (PBS containing 1% Triton X-100 and complete protease inhibitor cocktail tablets [Roche, Mannheim, Germany]) at 4 °C for 30 min.

Protease Inhibitor:

Article Title: CAEV Vif Hijacks ElonginB/C, CYPA and Cullin5 to Assemble the E3 Ubiquitin Ligase Complex Stepwise to Degrade oaA3Z2-Z3
Article Snippet: .. At 48 h post transfection, the HEK293T cells were harvested and washed twice with cold PBS, followed by dissociating in lysis buffer (PBS containing 1% Triton X-100 and complete protease inhibitor cocktail tablets [Roche, Mannheim, Germany]) at 4 °C for 30 min. .. The pre-cleared supernatants were collected by centrifugation at 10,000 g for 30 min at 4 °C and then mixed with anti-HA antibody conjugated agarose beads (cCatalog nNo.

Article Title: NADPH oxidase 2 inhibitors CPP11G and CPP11H attenuate endothelial cell inflammation vessel dysfunction and restore mouse hind-limb flow
Article Snippet: .. 4.6 Measurements H2 O2 production by Amplex Red Assay HAECs were collected in lysis buffer (Hank's Balanced Salt Solution with Complete Mini protease inhibitor and PhosStop phosphatase inhibitor from Roche), and were lysed by five freeze/thaw cycles and passed through a 30-gauge needle five times to disrupt cells. ..

Article Title: Identification and functional analysis of NOL7 nuclear and nucleolar localization signals
Article Snippet: .. Cells were pelleted by centrifugation and resuspended in lysis buffer (50 mM sodium phosphate, pH 7.4; 300 mM NaCl; 1% Triton X-100; Roche Complete EDTA-free protease inhibitor tablet). ..

Article Title: Knockdown of IKK1/2 Promotes Differentiation of Mouse Embryonic Stem Cells into Neuroectoderm at the Expense of Mesoderm
Article Snippet: .. Western Blot Cells were extracted in lysis-buffer (1 % SDS, 5 mM EDTA, complete protease inhibitor cocktail (Roche)) directly in the dish on ice. .. Equal amounts of protein extracts were separated on a 10 % SDS-PAGE and transferred to nitrocellulose membranes (PALL).

Article Title: Co-Regulation of NF-?B and Inflammasome-Mediated Inflammatory Responses by Myxoma Virus Pyrin Domain-Containing Protein M013
Article Snippet: .. Forty eight hours post transfection, cells were collected in 500 µl PBS, pelleted and then lysed by suspending in 20 µl lysis buffer (100 mM NaCl, 100 mM Tris, pH 8.0, 0.5% NP-40, containing 25 µl/ml Roche complete protease inhibitor). ..

Article Title: Expression, purification, crystallization and preliminary crystallographic analysis of MxiH, a subunit of the Shigella flexneri type III secretion system needle
Article Snippet: .. After ∼16 h, cells were harvested by centrifugation (15 min, 5000g , 277 K) and pellets were frozen at 193 K. Cell pellets were resuspended in lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl and Complete EDTA-free protease inhibitor cocktail, Roche) and lysed using an Emulsiflex-C5 Homogeniser (Glen Creston, UK). .. The resultant cell suspension was centrifuged (20 min, 20 000g , 277 K) and the soluble fraction was applied to a pre-charged HiTrap HP nickel-affinity column (HiTrap Chelating HP, Amersham Biosciences).

Article Title: Inhibition of HCV Replication by Oxysterol-Binding Protein-Related Protein 4 (ORP4) through Interaction with HCV NS5B and Alteration of Lipid Droplet Formation
Article Snippet: .. Cells were then washed with ice-cold PBS and lysed in 100 µl lysis buffer (1% Triton X-100 and 1X Roche protease inhibitor cocktail in PBS). .. The cell lysates were centrifuged at 12,000g for 20 min and the concentration of lysates was determined using a BioRad DC kit.

Article Title: A NUMB–EFA6B–ARF6 recycling route controls apically restricted cell protrusions and mesenchymal motility
Article Snippet: .. After 15 min of centrifugation at 1,200 rpm, pellets were suspended in lysis buffer (2× TBS, 0.5 mM EDTA, 10% glycerol, protease inhibitor cocktail [Roche; freshly added], and 1 mM DTT [freshly added]; 25 ml for 1-liter culture) or conserved at −80°C after washing in PBS once. ..

Article Title: Nuclear transport adapts to varying heat stress in a multistep mechanism
Article Snippet: .. Then, the cells were suspended in 500 µl lysis buffer (0.5% Triton X-100, 20 mM N -ethylmaleimide, and protease inhibitor cocktail [11873580001; Roche] in PBS− ) and rotated for 20 min at 4°C. .. After centrifugation, the supernatants were collected as soluble fractions.

Article Title: A two-hit mechanism causes cerebral cavernous malformations: complete inactivation of CCM1, CCM2 or CCM3 in affected endothelial cells
Article Snippet: .. Cell lysates were prepared with lysis buffer (1% Triton-X-100, 10 mm Tris pH 8, 1 mm EDTA, Roche complete EDTA-free protease inhibitor). .. For western blot analysis, 10 µg of tissue and cell lysates were loaded onto 10–20% SDS–PAGE gels, electroblotted onto nitrocellulose and probed with the polyclonal rabbit anti-human CCM3 antibody at a dilution of 1 µg/ml.

Cell Culture:

Article Title: Inhibition of HCV Replication by Oxysterol-Binding Protein-Related Protein 4 (ORP4) through Interaction with HCV NS5B and Alteration of Lipid Droplet Formation
Article Snippet: Forty-eight hours post transfection, the cell culture supernatants were collected and cell debris was removed by a brief centrifugation. .. Cells were then washed with ice-cold PBS and lysed in 100 µl lysis buffer (1% Triton X-100 and 1X Roche protease inhibitor cocktail in PBS).

Article Title: Reptin Regulates DNA Double Strand Breaks Repair in Human Hepatocellular Carcinoma
Article Snippet: Cells were washed with medium supplemented with 2 mM cysteine/methionine, and cultured for various times in this chase medium. .. Cells were then scraped in lysis buffer (5 PBS with 1% Triton X-100, Complete x1, Roche) and sonicated for 30 s on ice.

SDS Page:

Article Title: Identification and functional analysis of NOL7 nuclear and nucleolar localization signals
Article Snippet: Cells were pelleted by centrifugation and resuspended in lysis buffer (50 mM sodium phosphate, pH 7.4; 300 mM NaCl; 1% Triton X-100; Roche Complete EDTA-free protease inhibitor tablet). .. Fractions of approximately 300 μl were collected and tested for the presence of NOL7 by SDS-PAGE followed by silver staining and western blot using mouse α-V5 monoclonal antibody (Invitrogen, Carlsbad CA).

Article Title: Knockdown of IKK1/2 Promotes Differentiation of Mouse Embryonic Stem Cells into Neuroectoderm at the Expense of Mesoderm
Article Snippet: Western Blot Cells were extracted in lysis-buffer (1 % SDS, 5 mM EDTA, complete protease inhibitor cocktail (Roche)) directly in the dish on ice. .. Equal amounts of protein extracts were separated on a 10 % SDS-PAGE and transferred to nitrocellulose membranes (PALL).

Article Title: A two-hit mechanism causes cerebral cavernous malformations: complete inactivation of CCM1, CCM2 or CCM3 in affected endothelial cells
Article Snippet: Cell lysates were prepared with lysis buffer (1% Triton-X-100, 10 mm Tris pH 8, 1 mm EDTA, Roche complete EDTA-free protease inhibitor). .. For western blot analysis, 10 µg of tissue and cell lysates were loaded onto 10–20% SDS–PAGE gels, electroblotted onto nitrocellulose and probed with the polyclonal rabbit anti-human CCM3 antibody at a dilution of 1 µg/ml.

Imaging:

Article Title: A steep phosphoinositide bis-phosphate gradient forms during fungal filamentous growth
Article Snippet: Membranes were probed with anti-GFP polyclonal antibody (1:2,000; ) or S . cerevisiae anti-Cdc11 polyclonal antibody (sc-7170,1:200; Santa Cruz Biotechnology, Inc.), visualized by enhanced chemiluminescence (luminol-coumaric acid) on an imaging system (Las3000; Fujifilm). .. Wild-type or mutant cells (25 OD600 of exponentially growing cells) were harvested and lysed with glass beads and 300 µl of lysis buffer (25 mM KPO4 , 100 mM NaCl, 2 mM EDTA, and complete protease inhibitors; Roche).

Sonication:

Article Title: Nuclear transport adapts to varying heat stress in a multistep mechanism
Article Snippet: Then, the cells were suspended in 500 µl lysis buffer (0.5% Triton X-100, 20 mM N -ethylmaleimide, and protease inhibitor cocktail [11873580001; Roche] in PBS− ) and rotated for 20 min at 4°C. .. Remaining insoluble materials were resuspended in 500 µl of fresh lysis buffer and briefly sonicated.

Article Title: Reptin Regulates DNA Double Strand Breaks Repair in Human Hepatocellular Carcinoma
Article Snippet: .. Cells were then scraped in lysis buffer (5 PBS with 1% Triton X-100, Complete x1, Roche) and sonicated for 30 s on ice. ..

Recombinant:

Article Title: Expression, purification, crystallization and preliminary crystallographic analysis of MxiH, a subunit of the Shigella flexneri type III secretion system needle
Article Snippet: Expression and purification Recombinant forms of MxiHCΔ5 were produced and purified. .. After ∼16 h, cells were harvested by centrifugation (15 min, 5000g , 277 K) and pellets were frozen at 193 K. Cell pellets were resuspended in lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl and Complete EDTA-free protease inhibitor cocktail, Roche) and lysed using an Emulsiflex-C5 Homogeniser (Glen Creston, UK).

Amplified Luminescent Proximity Homogenous Assay:

Article Title: Co-Regulation of NF-?B and Inflammasome-Mediated Inflammatory Responses by Myxoma Virus Pyrin Domain-Containing Protein M013
Article Snippet: Paragraph title: AlphaScreen protein interaction assay ... Forty eight hours post transfection, cells were collected in 500 µl PBS, pelleted and then lysed by suspending in 20 µl lysis buffer (100 mM NaCl, 100 mM Tris, pH 8.0, 0.5% NP-40, containing 25 µl/ml Roche complete protease inhibitor).

Mutagenesis:

Article Title: A steep phosphoinositide bis-phosphate gradient forms during fungal filamentous growth
Article Snippet: .. Wild-type or mutant cells (25 OD600 of exponentially growing cells) were harvested and lysed with glass beads and 300 µl of lysis buffer (25 mM KPO4 , 100 mM NaCl, 2 mM EDTA, and complete protease inhibitors; Roche). .. After cell lysis, equivalent amounts of protein from each extract were centrifuged at 1,500g to remove intact cells and debris, resulting in total cell lysates.

Amplex Red Assay:

Article Title: NADPH oxidase 2 inhibitors CPP11G and CPP11H attenuate endothelial cell inflammation vessel dysfunction and restore mouse hind-limb flow
Article Snippet: .. 4.6 Measurements H2 O2 production by Amplex Red Assay HAECs were collected in lysis buffer (Hank's Balanced Salt Solution with Complete Mini protease inhibitor and PhosStop phosphatase inhibitor from Roche), and were lysed by five freeze/thaw cycles and passed through a 30-gauge needle five times to disrupt cells. ..

Size-exclusion Chromatography:

Article Title: Identification and functional analysis of NOL7 nuclear and nucleolar localization signals
Article Snippet: Cells were pelleted by centrifugation and resuspended in lysis buffer (50 mM sodium phosphate, pH 7.4; 300 mM NaCl; 1% Triton X-100; Roche Complete EDTA-free protease inhibitor tablet). .. Size-exclusion chromatography was performed using a 0.7 cm × 50 cm Econo-column (Bio-Rad, Hercules CA) that was packed with 5-100 kDA polyacrylamide beads (Bio-gel P-100, 45-90 μM, Bio-Rad, Hercules CA) according to manufacturer's instructions.

Labeling:

Article Title: Reptin Regulates DNA Double Strand Breaks Repair in Human Hepatocellular Carcinoma
Article Snippet: Paragraph title: Metabolic labeling ... Cells were then scraped in lysis buffer (5 PBS with 1% Triton X-100, Complete x1, Roche) and sonicated for 30 s on ice.

Purification:

Article Title: Identification and functional analysis of NOL7 nuclear and nucleolar localization signals
Article Snippet: For purification, approximately 1 × 108 cells were collected by trypsinization and washed twice with ice cold PBS. .. Cells were pelleted by centrifugation and resuspended in lysis buffer (50 mM sodium phosphate, pH 7.4; 300 mM NaCl; 1% Triton X-100; Roche Complete EDTA-free protease inhibitor tablet).

Article Title: Expression, purification, crystallization and preliminary crystallographic analysis of MxiH, a subunit of the Shigella flexneri type III secretion system needle
Article Snippet: Paragraph title: Expression and purification ... After ∼16 h, cells were harvested by centrifugation (15 min, 5000g , 277 K) and pellets were frozen at 193 K. Cell pellets were resuspended in lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl and Complete EDTA-free protease inhibitor cocktail, Roche) and lysed using an Emulsiflex-C5 Homogeniser (Glen Creston, UK).

Protein Purification:

Article Title: Identification and functional analysis of NOL7 nuclear and nucleolar localization signals
Article Snippet: Paragraph title: Protein Purification ... Cells were pelleted by centrifugation and resuspended in lysis buffer (50 mM sodium phosphate, pH 7.4; 300 mM NaCl; 1% Triton X-100; Roche Complete EDTA-free protease inhibitor tablet).

Lysis:

Article Title: CAEV Vif Hijacks ElonginB/C, CYPA and Cullin5 to Assemble the E3 Ubiquitin Ligase Complex Stepwise to Degrade oaA3Z2-Z3
Article Snippet: .. At 48 h post transfection, the HEK293T cells were harvested and washed twice with cold PBS, followed by dissociating in lysis buffer (PBS containing 1% Triton X-100 and complete protease inhibitor cocktail tablets [Roche, Mannheim, Germany]) at 4 °C for 30 min. .. The pre-cleared supernatants were collected by centrifugation at 10,000 g for 30 min at 4 °C and then mixed with anti-HA antibody conjugated agarose beads (cCatalog nNo.

Article Title: NADPH oxidase 2 inhibitors CPP11G and CPP11H attenuate endothelial cell inflammation vessel dysfunction and restore mouse hind-limb flow
Article Snippet: .. 4.6 Measurements H2 O2 production by Amplex Red Assay HAECs were collected in lysis buffer (Hank's Balanced Salt Solution with Complete Mini protease inhibitor and PhosStop phosphatase inhibitor from Roche), and were lysed by five freeze/thaw cycles and passed through a 30-gauge needle five times to disrupt cells. ..

Article Title: A steep phosphoinositide bis-phosphate gradient forms during fungal filamentous growth
Article Snippet: .. Wild-type or mutant cells (25 OD600 of exponentially growing cells) were harvested and lysed with glass beads and 300 µl of lysis buffer (25 mM KPO4 , 100 mM NaCl, 2 mM EDTA, and complete protease inhibitors; Roche). .. After cell lysis, equivalent amounts of protein from each extract were centrifuged at 1,500g to remove intact cells and debris, resulting in total cell lysates.

Article Title: Identification and functional analysis of NOL7 nuclear and nucleolar localization signals
Article Snippet: .. Cells were pelleted by centrifugation and resuspended in lysis buffer (50 mM sodium phosphate, pH 7.4; 300 mM NaCl; 1% Triton X-100; Roche Complete EDTA-free protease inhibitor tablet). ..

Article Title: Knockdown of IKK1/2 Promotes Differentiation of Mouse Embryonic Stem Cells into Neuroectoderm at the Expense of Mesoderm
Article Snippet: .. Western Blot Cells were extracted in lysis-buffer (1 % SDS, 5 mM EDTA, complete protease inhibitor cocktail (Roche)) directly in the dish on ice. .. Equal amounts of protein extracts were separated on a 10 % SDS-PAGE and transferred to nitrocellulose membranes (PALL).

Article Title: Co-Regulation of NF-?B and Inflammasome-Mediated Inflammatory Responses by Myxoma Virus Pyrin Domain-Containing Protein M013
Article Snippet: .. Forty eight hours post transfection, cells were collected in 500 µl PBS, pelleted and then lysed by suspending in 20 µl lysis buffer (100 mM NaCl, 100 mM Tris, pH 8.0, 0.5% NP-40, containing 25 µl/ml Roche complete protease inhibitor). ..

Article Title: Expression, purification, crystallization and preliminary crystallographic analysis of MxiH, a subunit of the Shigella flexneri type III secretion system needle
Article Snippet: .. After ∼16 h, cells were harvested by centrifugation (15 min, 5000g , 277 K) and pellets were frozen at 193 K. Cell pellets were resuspended in lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl and Complete EDTA-free protease inhibitor cocktail, Roche) and lysed using an Emulsiflex-C5 Homogeniser (Glen Creston, UK). .. The resultant cell suspension was centrifuged (20 min, 20 000g , 277 K) and the soluble fraction was applied to a pre-charged HiTrap HP nickel-affinity column (HiTrap Chelating HP, Amersham Biosciences).

Article Title: Inhibition of HCV Replication by Oxysterol-Binding Protein-Related Protein 4 (ORP4) through Interaction with HCV NS5B and Alteration of Lipid Droplet Formation
Article Snippet: .. Cells were then washed with ice-cold PBS and lysed in 100 µl lysis buffer (1% Triton X-100 and 1X Roche protease inhibitor cocktail in PBS). .. The cell lysates were centrifuged at 12,000g for 20 min and the concentration of lysates was determined using a BioRad DC kit.

Article Title: A NUMB–EFA6B–ARF6 recycling route controls apically restricted cell protrusions and mesenchymal motility
Article Snippet: .. After 15 min of centrifugation at 1,200 rpm, pellets were suspended in lysis buffer (2× TBS, 0.5 mM EDTA, 10% glycerol, protease inhibitor cocktail [Roche; freshly added], and 1 mM DTT [freshly added]; 25 ml for 1-liter culture) or conserved at −80°C after washing in PBS once. ..

Article Title: Nuclear transport adapts to varying heat stress in a multistep mechanism
Article Snippet: .. Then, the cells were suspended in 500 µl lysis buffer (0.5% Triton X-100, 20 mM N -ethylmaleimide, and protease inhibitor cocktail [11873580001; Roche] in PBS− ) and rotated for 20 min at 4°C. .. After centrifugation, the supernatants were collected as soluble fractions.

Article Title: A two-hit mechanism causes cerebral cavernous malformations: complete inactivation of CCM1, CCM2 or CCM3 in affected endothelial cells
Article Snippet: .. Cell lysates were prepared with lysis buffer (1% Triton-X-100, 10 mm Tris pH 8, 1 mm EDTA, Roche complete EDTA-free protease inhibitor). .. For western blot analysis, 10 µg of tissue and cell lysates were loaded onto 10–20% SDS–PAGE gels, electroblotted onto nitrocellulose and probed with the polyclonal rabbit anti-human CCM3 antibody at a dilution of 1 µg/ml.

Article Title: Reptin Regulates DNA Double Strand Breaks Repair in Human Hepatocellular Carcinoma
Article Snippet: .. Cells were then scraped in lysis buffer (5 PBS with 1% Triton X-100, Complete x1, Roche) and sonicated for 30 s on ice. ..

Silver Staining:

Article Title: Identification and functional analysis of NOL7 nuclear and nucleolar localization signals
Article Snippet: Cells were pelleted by centrifugation and resuspended in lysis buffer (50 mM sodium phosphate, pH 7.4; 300 mM NaCl; 1% Triton X-100; Roche Complete EDTA-free protease inhibitor tablet). .. Fractions of approximately 300 μl were collected and tested for the presence of NOL7 by SDS-PAGE followed by silver staining and western blot using mouse α-V5 monoclonal antibody (Invitrogen, Carlsbad CA).

Protein Interaction Assay:

Article Title: Co-Regulation of NF-?B and Inflammasome-Mediated Inflammatory Responses by Myxoma Virus Pyrin Domain-Containing Protein M013
Article Snippet: Paragraph title: AlphaScreen protein interaction assay ... Forty eight hours post transfection, cells were collected in 500 µl PBS, pelleted and then lysed by suspending in 20 µl lysis buffer (100 mM NaCl, 100 mM Tris, pH 8.0, 0.5% NP-40, containing 25 µl/ml Roche complete protease inhibitor).

Plasmid Preparation:

Article Title: Co-Regulation of NF-?B and Inflammasome-Mediated Inflammatory Responses by Myxoma Virus Pyrin Domain-Containing Protein M013
Article Snippet: On the other hand, expression from the pANT7_cGST plasmid is under the control of T7 RNA polymerase promoter and expressed proteins are fused at their C-terminal with a glutathione-S-transferase (GST) tag. .. Forty eight hours post transfection, cells were collected in 500 µl PBS, pelleted and then lysed by suspending in 20 µl lysis buffer (100 mM NaCl, 100 mM Tris, pH 8.0, 0.5% NP-40, containing 25 µl/ml Roche complete protease inhibitor).

Article Title: Expression, purification, crystallization and preliminary crystallographic analysis of MxiH, a subunit of the Shigella flexneri type III secretion system needle
Article Snippet: The DNA fragment of the mxiH gene encoding residues 1–78 was produced as described previously (Kenjale et al. , 2005 ) and subcloned into the pET22b vector. .. After ∼16 h, cells were harvested by centrifugation (15 min, 5000g , 277 K) and pellets were frozen at 193 K. Cell pellets were resuspended in lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl and Complete EDTA-free protease inhibitor cocktail, Roche) and lysed using an Emulsiflex-C5 Homogeniser (Glen Creston, UK).

Homogenization:

Article Title: A two-hit mechanism causes cerebral cavernous malformations: complete inactivation of CCM1, CCM2 or CCM3 in affected endothelial cells
Article Snippet: Mouse tissues were homogenized in an iso-osmotic homogenization buffer (0.25 m sucrose, 10 mm Tris pH 7,2, 1 mm EDTA, Roche complete EDTA-free protease inhibitor) by five passes with 1000 U/min using a Potter S homogenizer ( ). .. Cell lysates were prepared with lysis buffer (1% Triton-X-100, 10 mm Tris pH 8, 1 mm EDTA, Roche complete EDTA-free protease inhibitor).

Quantitation Assay:

Article Title: A steep phosphoinositide bis-phosphate gradient forms during fungal filamentous growth
Article Snippet: Paragraph title: Immunoblot analyses and PI(4,5)P2 quantitation ... Wild-type or mutant cells (25 OD600 of exponentially growing cells) were harvested and lysed with glass beads and 300 µl of lysis buffer (25 mM KPO4 , 100 mM NaCl, 2 mM EDTA, and complete protease inhibitors; Roche).

Produced:

Article Title: Expression, purification, crystallization and preliminary crystallographic analysis of MxiH, a subunit of the Shigella flexneri type III secretion system needle
Article Snippet: The DNA fragment of the mxiH gene encoding residues 1–78 was produced as described previously (Kenjale et al. , 2005 ) and subcloned into the pET22b vector. .. After ∼16 h, cells were harvested by centrifugation (15 min, 5000g , 277 K) and pellets were frozen at 193 K. Cell pellets were resuspended in lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl and Complete EDTA-free protease inhibitor cocktail, Roche) and lysed using an Emulsiflex-C5 Homogeniser (Glen Creston, UK).

Article Title: A NUMB–EFA6B–ARF6 recycling route controls apically restricted cell protrusions and mesenchymal motility
Article Snippet: Proteins were finally produced from 500 ml of H5 cells seeded at a concentration of 0.5 × 106 cells/ml and infected with 12.5 ml of V2 virus. .. After 15 min of centrifugation at 1,200 rpm, pellets were suspended in lysis buffer (2× TBS, 0.5 mM EDTA, 10% glycerol, protease inhibitor cocktail [Roche; freshly added], and 1 mM DTT [freshly added]; 25 ml for 1-liter culture) or conserved at −80°C after washing in PBS once.

Concentration Assay:

Article Title: Co-Regulation of NF-?B and Inflammasome-Mediated Inflammatory Responses by Myxoma Virus Pyrin Domain-Containing Protein M013
Article Snippet: Forty eight hours post transfection, cells were collected in 500 µl PBS, pelleted and then lysed by suspending in 20 µl lysis buffer (100 mM NaCl, 100 mM Tris, pH 8.0, 0.5% NP-40, containing 25 µl/ml Roche complete protease inhibitor). .. Five µl of anti-GST acceptor beads (PerkinElmer, 6760603C), diluted 50-fold (20 µg/ml final concentration of beads in assay) in 0.1% BSA/PBS assay buffer, and 5 µl of cell extract were mixed to a final volume of 20 µl with 0.1% BSA/PBS assay buffer, and incubated at room temperature for 1.5 hour.

Article Title: Inhibition of HCV Replication by Oxysterol-Binding Protein-Related Protein 4 (ORP4) through Interaction with HCV NS5B and Alteration of Lipid Droplet Formation
Article Snippet: Cells were then washed with ice-cold PBS and lysed in 100 µl lysis buffer (1% Triton X-100 and 1X Roche protease inhibitor cocktail in PBS). .. The cell lysates were centrifuged at 12,000g for 20 min and the concentration of lysates was determined using a BioRad DC kit.

Article Title: A NUMB–EFA6B–ARF6 recycling route controls apically restricted cell protrusions and mesenchymal motility
Article Snippet: Proteins were finally produced from 500 ml of H5 cells seeded at a concentration of 0.5 × 106 cells/ml and infected with 12.5 ml of V2 virus. .. After 15 min of centrifugation at 1,200 rpm, pellets were suspended in lysis buffer (2× TBS, 0.5 mM EDTA, 10% glycerol, protease inhibitor cocktail [Roche; freshly added], and 1 mM DTT [freshly added]; 25 ml for 1-liter culture) or conserved at −80°C after washing in PBS once.

Fractionation:

Article Title: Nuclear transport adapts to varying heat stress in a multistep mechanism
Article Snippet: Paragraph title: Fractionation ... Then, the cells were suspended in 500 µl lysis buffer (0.5% Triton X-100, 20 mM N -ethylmaleimide, and protease inhibitor cocktail [11873580001; Roche] in PBS− ) and rotated for 20 min at 4°C.

Amplex Red Cholesterol Assay:

Article Title: Inhibition of HCV Replication by Oxysterol-Binding Protein-Related Protein 4 (ORP4) through Interaction with HCV NS5B and Alteration of Lipid Droplet Formation
Article Snippet: Cholesterol assay Cholesterol levels in cell culture supernatants and cells were measured using an Amplex Red Cholesterol Assay Kit (Invitrogen). .. Cells were then washed with ice-cold PBS and lysed in 100 µl lysis buffer (1% Triton X-100 and 1X Roche protease inhibitor cocktail in PBS).

Staining:

Article Title: Reptin Regulates DNA Double Strand Breaks Repair in Human Hepatocellular Carcinoma
Article Snippet: Cells were then scraped in lysis buffer (5 PBS with 1% Triton X-100, Complete x1, Roche) and sonicated for 30 s on ice. .. The gel was stained with Coomassie blue, destained in water, impregnated with ENHANCE, dried, and exposed to Amersham XP film at -80°C.

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  • 95
    Roche flag lysis buffer
    In vitro RNA binding activity of full-length HBV with mutant T3 and RT1 motifs (a) Accumulation of HBV P mutants. HBV P derivatives were expressed in transfected 293T cells, immunoprecipitated with <t>anti-FLAG</t> antibodies, resolved by SDS-PAGE, and detected by western blotting using the <t>M2</t> anti-FLAG antibody; the exposure of the left gel was shorter to limit saturation of the more intense bands. The position of HBV P (P) and the antibody heavy chain (HC) are indicated. * denotes the position of an N-terminal fragment of the 3xFLAG-tagged wild-type P. (b) The immunoaffinity-purified HBV P derivatives were incubated with 32 P-labeled wild-type Hε or mutant Hε-dB RNA and co-precipitated products were resolved by SDS–PAGE. Input representing 0.5% of the indicated ε RNA added to each binding reaction mixture is in lanes 10, 11, 20, and 21. (c) Bound 32 P-labeled ε RNA signals were quantified via phosphorimaging and compared to the binding of wild-type P to Hε RNA. The data represent the mean ± one standard deviation from at least three independent experiments.
    Flag Lysis Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 95/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Roche sds lysis buffer
    LRRK2 forms kinase-sensitive high molecular weight conformations. A , cell pellets derived from four lymphoblast cell lines (labeled Lymph1 to -4 ) were split equally for lysis by freeze/thaw cycles directly in <t>PBS</t> or lysis with 1% <t>SDS</t> and PBS with sonication,
    Sds Lysis Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sds lysis buffer/product/Roche
    Average 99 stars, based on 25 article reviews
    Price from $9.99 to $1999.99
    sds lysis buffer - by Bioz Stars, 2020-04
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    94
    Roche nondenaturing np 40 lysis buffer
    Western blot analysis of pyrin S205 dephosphorylation in BMDMs. LPS-primed BMDMs were left uninfected, infected with Y. pseudotuberculosis strain IP6ΔM harboring an empty vector or IP6ΔM/pYopE or intoxicated with TcdB for 90 min. (A and B) Lysates were processed with <t>nondenaturing</t> <t>NP-40</t> lysis buffer and separated into soluble and insoluble fractions by centrifugation. (C) Lysates were processed with denaturing RIPA lysis buffer, and the soluble fractions were collected. Samples were subjected to Western blotting using antibodies to pSer205 of pyrin, total pyrin, or β-actin.
    Nondenaturing Np 40 Lysis 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
    https://www.bioz.com/result/nondenaturing np 40 lysis buffer/product/Roche
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    nondenaturing np 40 lysis buffer - by Bioz Stars, 2020-04
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    99
    Roche np 40 lysis buffer
    CsCl density gradient analysis of hepatitis B viral particles. (A and B) CsCl density gradient analysis of viral particles in patient sera. One hundred-microliter volumes of serum mixture from patients 37, 38, 14, and 35 (25 μl each) and 100 μl serum from patient 17 were separated by CsCl density gradient centrifugation (2 ml of 1.18 g/cm 3 CsCl solution in the upper layer and 2.9 ml of 1.33 g/cm 3 CsCl solution in the lower layer). Viral DNA in each fraction was extracted and detected by Southern blotting. (C to G) CsCl density gradient analysis of viral particles treated with detergent or anti-HBcAg antibody (Ab). Concentrated HepAD38 cell culture supernatant (250 μl each) (via ultrafiltration) was either mixed with anti-HBcAg antibody (10 μl) followed by incubation without (C) or with <t>NP-40</t> (final concentration, 1%) (D) for 1 h at room temperature and 4 h on ice or treated with only NP-40 (G) and then fractionated by CsCl density gradient ultracentrifugation. Sera from CHB patient 46 either left untreated (E) or treated with NP-40 (final concentration, 1%) (F) were fractionated by CsCl density gradient ultracentrifugation. Viral DNA in each fraction was extracted and subjected to Southern blot analyses.
    Np 40 Lysis Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 137 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    In vitro RNA binding activity of full-length HBV with mutant T3 and RT1 motifs (a) Accumulation of HBV P mutants. HBV P derivatives were expressed in transfected 293T cells, immunoprecipitated with anti-FLAG antibodies, resolved by SDS-PAGE, and detected by western blotting using the M2 anti-FLAG antibody; the exposure of the left gel was shorter to limit saturation of the more intense bands. The position of HBV P (P) and the antibody heavy chain (HC) are indicated. * denotes the position of an N-terminal fragment of the 3xFLAG-tagged wild-type P. (b) The immunoaffinity-purified HBV P derivatives were incubated with 32 P-labeled wild-type Hε or mutant Hε-dB RNA and co-precipitated products were resolved by SDS–PAGE. Input representing 0.5% of the indicated ε RNA added to each binding reaction mixture is in lanes 10, 11, 20, and 21. (c) Bound 32 P-labeled ε RNA signals were quantified via phosphorimaging and compared to the binding of wild-type P to Hε RNA. The data represent the mean ± one standard deviation from at least three independent experiments.

    Journal: Journal of viral hepatitis

    Article Title: Sequences in the terminal protein and reverse transcriptase domains of the Hepatitis B Virus polymerase contribute to RNA binding and encapsidation

    doi: 10.1111/jvh.12225

    Figure Lengend Snippet: In vitro RNA binding activity of full-length HBV with mutant T3 and RT1 motifs (a) Accumulation of HBV P mutants. HBV P derivatives were expressed in transfected 293T cells, immunoprecipitated with anti-FLAG antibodies, resolved by SDS-PAGE, and detected by western blotting using the M2 anti-FLAG antibody; the exposure of the left gel was shorter to limit saturation of the more intense bands. The position of HBV P (P) and the antibody heavy chain (HC) are indicated. * denotes the position of an N-terminal fragment of the 3xFLAG-tagged wild-type P. (b) The immunoaffinity-purified HBV P derivatives were incubated with 32 P-labeled wild-type Hε or mutant Hε-dB RNA and co-precipitated products were resolved by SDS–PAGE. Input representing 0.5% of the indicated ε RNA added to each binding reaction mixture is in lanes 10, 11, 20, and 21. (c) Bound 32 P-labeled ε RNA signals were quantified via phosphorimaging and compared to the binding of wild-type P to Hε RNA. The data represent the mean ± one standard deviation from at least three independent experiments.

    Article Snippet: The FLAG lysis buffer was removed from aliquots of P-bound M2 beads, and then aliquots of beads were incubated with 0.5 μg 32 P-labeled ε RNAs in radioimmunoprecipitation assay (RIPA) buffer [50 mM Tris (pH 7.0), 150 mM NaCl, 1 mM EDTA, 0.05% NP-40] with 1× complete protease inhibitor cocktail (Roche), 2 mM DTT, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 U/μl RNasin Plus RNase inhibitor (Promega) ( ).

    Techniques: In Vitro, RNA Binding Assay, Activity Assay, Mutagenesis, Transfection, Immunoprecipitation, SDS Page, Western Blot, Purification, Incubation, Labeling, Binding Assay, Standard Deviation

    LRRK2 forms kinase-sensitive high molecular weight conformations. A , cell pellets derived from four lymphoblast cell lines (labeled Lymph1 to -4 ) were split equally for lysis by freeze/thaw cycles directly in PBS or lysis with 1% SDS and PBS with sonication,

    Journal: The Journal of Biological Chemistry

    Article Title: Dependence of Leucine-rich Repeat Kinase 2 (LRRK2) Kinase Activity on Dimerization *

    doi: 10.1074/jbc.M109.025437

    Figure Lengend Snippet: LRRK2 forms kinase-sensitive high molecular weight conformations. A , cell pellets derived from four lymphoblast cell lines (labeled Lymph1 to -4 ) were split equally for lysis by freeze/thaw cycles directly in PBS or lysis with 1% SDS and PBS with sonication,

    Article Snippet: Cell pellets were resuspended with either native lysis buffer (phosphate-buffered saline (PBS), pH 7.4, 1× protease and phosphatase inhibitors (Roche Applied Science)), lysed by four cycles of freezing and thawing, and analyzed on a 3–12% bis-tris Blue native polyacrylamide gel (Invitrogen) or lysates combined with SDS-lysis buffer (PBS, pH 7.4, 1% SDS, 1× protease and phosphatase inhibitors (Roche Applied Science)) and sonicated with 10% power for 10 s on a Branson dismembranator and analyzed on a 7% Tris acetate SDS gel (Invitrogen).

    Techniques: Molecular Weight, Derivative Assay, Labeling, Lysis, Sonication

    Western blot analysis of pyrin S205 dephosphorylation in BMDMs. LPS-primed BMDMs were left uninfected, infected with Y. pseudotuberculosis strain IP6ΔM harboring an empty vector or IP6ΔM/pYopE or intoxicated with TcdB for 90 min. (A and B) Lysates were processed with nondenaturing NP-40 lysis buffer and separated into soluble and insoluble fractions by centrifugation. (C) Lysates were processed with denaturing RIPA lysis buffer, and the soluble fractions were collected. Samples were subjected to Western blotting using antibodies to pSer205 of pyrin, total pyrin, or β-actin.

    Journal: Infection and Immunity

    Article Title: Characterization of Pyrin Dephosphorylation and Inflammasome Activation in Macrophages as Triggered by the Yersinia Effectors YopE and YopT

    doi: 10.1128/IAI.00822-18

    Figure Lengend Snippet: Western blot analysis of pyrin S205 dephosphorylation in BMDMs. LPS-primed BMDMs were left uninfected, infected with Y. pseudotuberculosis strain IP6ΔM harboring an empty vector or IP6ΔM/pYopE or intoxicated with TcdB for 90 min. (A and B) Lysates were processed with nondenaturing NP-40 lysis buffer and separated into soluble and insoluble fractions by centrifugation. (C) Lysates were processed with denaturing RIPA lysis buffer, and the soluble fractions were collected. Samples were subjected to Western blotting using antibodies to pSer205 of pyrin, total pyrin, or β-actin.

    Article Snippet: To obtain cell lysates, BMDMs were lysed using nondenaturing NP-40 lysis buffer (1% NP-40, 150 mM NaCl, and 50 mM Tris-HCl, pH 8.0) or denaturing RIPA buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid [DOC], and 0.1% SDS) with protease inhibitor cocktail (Roche).

    Techniques: Western Blot, De-Phosphorylation Assay, Infection, Plasmid Preparation, Lysis, Centrifugation

    CsCl density gradient analysis of hepatitis B viral particles. (A and B) CsCl density gradient analysis of viral particles in patient sera. One hundred-microliter volumes of serum mixture from patients 37, 38, 14, and 35 (25 μl each) and 100 μl serum from patient 17 were separated by CsCl density gradient centrifugation (2 ml of 1.18 g/cm 3 CsCl solution in the upper layer and 2.9 ml of 1.33 g/cm 3 CsCl solution in the lower layer). Viral DNA in each fraction was extracted and detected by Southern blotting. (C to G) CsCl density gradient analysis of viral particles treated with detergent or anti-HBcAg antibody (Ab). Concentrated HepAD38 cell culture supernatant (250 μl each) (via ultrafiltration) was either mixed with anti-HBcAg antibody (10 μl) followed by incubation without (C) or with NP-40 (final concentration, 1%) (D) for 1 h at room temperature and 4 h on ice or treated with only NP-40 (G) and then fractionated by CsCl density gradient ultracentrifugation. Sera from CHB patient 46 either left untreated (E) or treated with NP-40 (final concentration, 1%) (F) were fractionated by CsCl density gradient ultracentrifugation. Viral DNA in each fraction was extracted and subjected to Southern blot analyses.

    Journal: Journal of Virology

    Article Title: Extracellular Hepatitis B Virus RNAs Are Heterogeneous in Length and Circulate as Capsid-Antibody Complexes in Addition to Virions in Chronic Hepatitis B Patients

    doi: 10.1128/JVI.00798-18

    Figure Lengend Snippet: CsCl density gradient analysis of hepatitis B viral particles. (A and B) CsCl density gradient analysis of viral particles in patient sera. One hundred-microliter volumes of serum mixture from patients 37, 38, 14, and 35 (25 μl each) and 100 μl serum from patient 17 were separated by CsCl density gradient centrifugation (2 ml of 1.18 g/cm 3 CsCl solution in the upper layer and 2.9 ml of 1.33 g/cm 3 CsCl solution in the lower layer). Viral DNA in each fraction was extracted and detected by Southern blotting. (C to G) CsCl density gradient analysis of viral particles treated with detergent or anti-HBcAg antibody (Ab). Concentrated HepAD38 cell culture supernatant (250 μl each) (via ultrafiltration) was either mixed with anti-HBcAg antibody (10 μl) followed by incubation without (C) or with NP-40 (final concentration, 1%) (D) for 1 h at room temperature and 4 h on ice or treated with only NP-40 (G) and then fractionated by CsCl density gradient ultracentrifugation. Sera from CHB patient 46 either left untreated (E) or treated with NP-40 (final concentration, 1%) (F) were fractionated by CsCl density gradient ultracentrifugation. Viral DNA in each fraction was extracted and subjected to Southern blot analyses.

    Article Snippet: To isolate intracellular capsid-associated viral RNA, HepAD38 cells were lysed in NP-40 lysis buffer (50 mM Tris-Cl [pH 7.8], 1 mM EDTA, 1% NP-40), and cytoplasmic lysates were incubated with CaCl2 (final concentration, 5 mM) and micrococcal nuclease (MNase) (Roche) (final concentration, 15 U/ml) at 37°C for 1 h to remove nucleic acids outside nucleocapsids.

    Techniques: Gradient Centrifugation, Southern Blot, Cell Culture, Incubation, Concentration Assay