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Roche radioimmuno precipitation assay buffer
Radioimmuno Precipitation Assay Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 90/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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radioimmuno precipitation assay buffer - by Bioz Stars, 2020-03
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Centrifugation:

Article Title: Oncogenic Ras/Src cooperativity in pancreatic neoplasia
Article Snippet: Both tissues and tumor cell lines were lysed in a modified radioimmuno precipitation assay buffer containing 50 m m Tris pH 7.5 at 4°C, 150 m m NaCl, 1 m m EDTA, 50 m m NaF, 5 m m sodium pyrophosphate, 10 m m β-glycerophosphate, 1% NP-40, 1 m m Na3VO4, 0.25% sodium deoxycholate, 0.1% SDS, phosphatase and protease inhibitors (Roche, Indianapolis, IN, USA). .. Lysates were cleared by centrifugation and the protein concentration of the cleared lysate was determined by the bicinchoninic acid method.

Article Title: Glycosylphosphatidyl Inositol-anchored Proteins and fyn Kinase Assemble in Noncaveolar Plasma Membrane Microdomains Defined by Reggie-1 and -2
Article Snippet: Two methods were used: 1) Rat brain tissue, DRGs, and pelleted cells were homogenized on ice in modified radioimmuno precipitation-assay buffer ( ) containing 1% NP-40 (Roche Molecular Biochemicals), 0.5% sodium deoxycholate (Sigma), and 0.2% SDS (Sigma) as well as a protease inhibitor cocktail (Complete Mini; Roche Molecular Biochemicals, Mannheim, Germany). .. The homogenates were incubated on ice and insoluble fractions removed by centrifugation.

BIA-KA:

Article Title: Epigenetics of programmed obesity: alteration in IUGR rat hepatic IGF1 mRNA expression and histone structure in rapid vs. delayed postnatal catch-up growth
Article Snippet: Protein was extracted from day 1 and adult liver using a radioimmuno precipitation assay buffer that contained a protease inhibitor cocktail (Roche, Manheim, Germany). .. Protein was extracted from day 1 and adult liver using a radioimmuno precipitation assay buffer that contained a protease inhibitor cocktail (Roche, Manheim, Germany).

Incubation:

Article Title: Highly Conserved Cysteines within the Ly6 Domain of GPIHBP1 Are Crucial for the Binding of Lipoprotein Lipase *
Article Snippet: .. At the end of the incubation period, the cells were washed six times in ice-cold PBS containing 1 m m MgCl2 and 1 m m CaCl2 , and cell extracts were collected in radioimmuno precipitation assay buffer (PBS containing 1% Triton X-100, 0.5% deoxycholate, and 0.02% SDS) along with complete mini EDTA-free protease inhibitors (Roche Applied Science). .. The amounts of LPL and GPIHBP1 in cell extracts were assessed by Western blotting.

Article Title: Highly Conserved Cysteines within the Ly6 Domain of GPIHBP1 Are Crucial for the Binding of Lipoprotein Lipase *
Article Snippet: .. At the end of the incubation, the medium was harvested, and cell extracts were collected in radioimmuno precipitation assay buffer containing complete mini EDTA-free protease inhibitors (Roche Applied Science). .. In other experiments, we assessed the amount of GPIHBP1 expression on the surface of cells compared with the total amount of GPIHBP1 within cells.

Article Title: Highly Conserved Cysteines within the Ly6 Domain of GPIHBP1 Are Crucial for the Binding of Lipoprotein Lipase *
Article Snippet: .. At the end of the incubation period, the cells were washed six times in ice-cold PBS/Mg/Ca, and the cell extracts were collected in radioimmuno precipitation assay buffer containing complete mini EDTA-free protease inhibitors (Roche Applied Science). .. To judge the amount of GPIHBP1 on the cell surface, we performed Western blots on cell extracts with an IRdye680-conjugated donkey anti-goat IgG (Li-Cor; 1:800).

Article Title: Cyclical stretch induces structural changes in atrial myocytes
Article Snippet: Western blotting Proteins from whole cell lysates were isolated using radioimmuno-precipitation assay buffer [0.5% sodium deoxycholate, 0.1% sodium dodecylsulfate (SDS) and 1% Igepal ca-630 in TBS] supplemented with a protease inhibitor cocktail (Roche Diagnostics Corp., Indianapolis, IN, USA), a phosphatase inhibitor cocktail, PMSF (1 mmol/l; Roche Diagnostics Corp.) and sodium vanadate (15 mmol/l). .. Blots were incubated overnight with antibodies against either phospho-Erk (E-4), troponin T-C (C-19) (both Santa Cruz Biotechnology), troponin I-C (MF4; Fitzgerald Industries International, Acton, MA, USA), phospho-Akt (Ser473) (D9E), phospho-p38 (Thr180/Tyr182; both Cell Signaling Technology, Danvers, MA, USA) or microtubule-associated protein light chain 3 (LC3; MBL, Naka-ku Nagoya, Japan) diluted in either 5% milk or BSA in TBST.

Article Title: Glycosylphosphatidyl Inositol-anchored Proteins and fyn Kinase Assemble in Noncaveolar Plasma Membrane Microdomains Defined by Reggie-1 and -2
Article Snippet: Two methods were used: 1) Rat brain tissue, DRGs, and pelleted cells were homogenized on ice in modified radioimmuno precipitation-assay buffer ( ) containing 1% NP-40 (Roche Molecular Biochemicals), 0.5% sodium deoxycholate (Sigma), and 0.2% SDS (Sigma) as well as a protease inhibitor cocktail (Complete Mini; Roche Molecular Biochemicals, Mannheim, Germany). .. The homogenates were incubated on ice and insoluble fractions removed by centrifugation.

Expressing:

Article Title: Highly Conserved Cysteines within the Ly6 Domain of GPIHBP1 Are Crucial for the Binding of Lipoprotein Lipase *
Article Snippet: At the end of the incubation, the medium was harvested, and cell extracts were collected in radioimmuno precipitation assay buffer containing complete mini EDTA-free protease inhibitors (Roche Applied Science). .. In other experiments, we assessed the amount of GPIHBP1 expression on the surface of cells compared with the total amount of GPIHBP1 within cells.

Bradford Assay:

Article Title: Optimisation of Intestinal Fibrosis and Survival in the Mouse S. Typhimurium Model for Anti-fibrotic Drug Discovery and Preclinical Applications
Article Snippet: .. Whole tissue was pulverized under liquid nitrogen and lysed in ice-cold radioimmuno precipitation assay buffer [RIPA buffer] with a cocktail of proteinase inhibitors [Roche, Indianapolis, IN]., Protein content was determined using a modified Bradford assay kit [BioRad, Hercules, CA]. .. Total protein was separated by sodium dodecyl sulfate [SDS] polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride [PVDF] membranes [Amersham Biosciences, Piscataway, NJ].

Modification:

Article Title: Oncogenic Ras/Src cooperativity in pancreatic neoplasia
Article Snippet: .. Both tissues and tumor cell lines were lysed in a modified radioimmuno precipitation assay buffer containing 50 m m Tris pH 7.5 at 4°C, 150 m m NaCl, 1 m m EDTA, 50 m m NaF, 5 m m sodium pyrophosphate, 10 m m β-glycerophosphate, 1% NP-40, 1 m m Na3VO4, 0.25% sodium deoxycholate, 0.1% SDS, phosphatase and protease inhibitors (Roche, Indianapolis, IN, USA). .. Lysates were cleared by centrifugation and the protein concentration of the cleared lysate was determined by the bicinchoninic acid method.

Article Title: Optimisation of Intestinal Fibrosis and Survival in the Mouse S. Typhimurium Model for Anti-fibrotic Drug Discovery and Preclinical Applications
Article Snippet: .. Whole tissue was pulverized under liquid nitrogen and lysed in ice-cold radioimmuno precipitation assay buffer [RIPA buffer] with a cocktail of proteinase inhibitors [Roche, Indianapolis, IN]., Protein content was determined using a modified Bradford assay kit [BioRad, Hercules, CA]. .. Total protein was separated by sodium dodecyl sulfate [SDS] polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride [PVDF] membranes [Amersham Biosciences, Piscataway, NJ].

Article Title: Glycosylphosphatidyl Inositol-anchored Proteins and fyn Kinase Assemble in Noncaveolar Plasma Membrane Microdomains Defined by Reggie-1 and -2
Article Snippet: .. Two methods were used: 1) Rat brain tissue, DRGs, and pelleted cells were homogenized on ice in modified radioimmuno precipitation-assay buffer ( ) containing 1% NP-40 (Roche Molecular Biochemicals), 0.5% sodium deoxycholate (Sigma), and 0.2% SDS (Sigma) as well as a protease inhibitor cocktail (Complete Mini; Roche Molecular Biochemicals, Mannheim, Germany). .. The homogenates were incubated on ice and insoluble fractions removed by centrifugation.

Western Blot:

Article Title: Oncogenic Ras/Src cooperativity in pancreatic neoplasia
Article Snippet: Both tissues and tumor cell lines were lysed in a modified radioimmuno precipitation assay buffer containing 50 m m Tris pH 7.5 at 4°C, 150 m m NaCl, 1 m m EDTA, 50 m m NaF, 5 m m sodium pyrophosphate, 10 m m β-glycerophosphate, 1% NP-40, 1 m m Na3VO4, 0.25% sodium deoxycholate, 0.1% SDS, phosphatase and protease inhibitors (Roche, Indianapolis, IN, USA). .. Immunoblots were performed using the following antibodies: pSFK-Y418 (Cell Signaling), Erk2, Fyn (Santa Cruz), Yes (BD Biosciences, San Diego, CA, USA), Src, pY-4G10 (Millipore, Billerica, MA, USA).

Article Title: Epigenetics of programmed obesity: alteration in IUGR rat hepatic IGF1 mRNA expression and histone structure in rapid vs. delayed postnatal catch-up growth
Article Snippet: Paragraph title: Western blot. ... Protein was extracted from day 1 and adult liver using a radioimmuno precipitation assay buffer that contained a protease inhibitor cocktail (Roche, Manheim, Germany).

Article Title: Highly Conserved Cysteines within the Ly6 Domain of GPIHBP1 Are Crucial for the Binding of Lipoprotein Lipase *
Article Snippet: At the end of the incubation period, the cells were washed six times in ice-cold PBS containing 1 m m MgCl2 and 1 m m CaCl2 , and cell extracts were collected in radioimmuno precipitation assay buffer (PBS containing 1% Triton X-100, 0.5% deoxycholate, and 0.02% SDS) along with complete mini EDTA-free protease inhibitors (Roche Applied Science). .. The amounts of LPL and GPIHBP1 in cell extracts were assessed by Western blotting.

Article Title: Loss of giant obscurins from breast epithelium promotes epithelial-to-mesenchymal transition, tumorigenicity and metastasis
Article Snippet: Paragraph title: Generation of protein lysates and western blotting ... Tissue homogenates were prepared on ice with a hand-homogenizer in radioimmuno-precipitation assay buffer supplemented with a cocktail of protease inhibitors (Roche, Mannheim, Germany) and phosphatase inhibitors (200 nM Imidazole, 100mM Sodium Flouride, 115 mM Sodium Molybdate, 100 mM Sodium Orthovanadate, 400 mM Sodium Tartrate Dihydrate, 100 mM β-Glycerophosphate, 100 mM Sodium Pyrophosphate and 10 mM EGTA), as previously described.

Article Title: Highly Conserved Cysteines within the Ly6 Domain of GPIHBP1 Are Crucial for the Binding of Lipoprotein Lipase *
Article Snippet: At the end of the incubation period, the cells were washed six times in ice-cold PBS/Mg/Ca, and the cell extracts were collected in radioimmuno precipitation assay buffer containing complete mini EDTA-free protease inhibitors (Roche Applied Science). .. To judge the amount of GPIHBP1 on the cell surface, we performed Western blots on cell extracts with an IRdye680-conjugated donkey anti-goat IgG (Li-Cor; 1:800).

Article Title: Adapted HCV JFH1 variant is capable of accommodating a large foreign gene insert and allows lower level HCV replication and viral production
Article Snippet: Paragraph title: Western blot analysis ... The HCV-transfected Huh 7.5 cells were lysed in a radioimmuno-precipitation assay buffer (50 mM Tris-HCl, pH 7.5, 150 mM sodium chloride, 1% Nonidet P40, 0.5% sodium deoxycholate) containing a cocktail of proteinase inhibitors (Roche).

Article Title: TGF-? receptor II in epithelia versus mesenchyme plays distinct roles in the developing lung
Article Snippet: Paragraph title: Western blot ... Briefly, fresh lung tissues were lysed on ice in radioimmuno precipitation assay buffer containing 1 mM phenyl-methyl sulphonyl fluoride, protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland) and 1 mM sodium orthovanadate.

Article Title: Adapted HCV JFH1 variant is capable of accommodating a large foreign gene insert and allows lower level HCV replication and viral production
Article Snippet: .. Western blot analysis The HCV-transfected Huh 7.5 cells were lysed in a radioimmuno-precipitation assay buffer (50 mM Tris-HCl, pH 7.5, 150 mM sodium chloride, 1% Nonidet P40, 0.5% sodium deoxycholate) containing a cocktail of proteinase inhibitors (Roche). ..

Article Title: Cyclical stretch induces structural changes in atrial myocytes
Article Snippet: .. Western blotting Proteins from whole cell lysates were isolated using radioimmuno-precipitation assay buffer [0.5% sodium deoxycholate, 0.1% sodium dodecylsulfate (SDS) and 1% Igepal ca-630 in TBS] supplemented with a protease inhibitor cocktail (Roche Diagnostics Corp., Indianapolis, IN, USA), a phosphatase inhibitor cocktail, PMSF (1 mmol/l; Roche Diagnostics Corp.) and sodium vanadate (15 mmol/l). .. SDS sample buffer was added and samples were denaturized by heat at 99°C for 5 min. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel, transferred to PVDF membranes (Bio-Rad) and blocked with 5% milk in 0.1% tween in TBS (TBST).

Electroporation:

Article Title: Highly Conserved Cysteines within the Ly6 Domain of GPIHBP1 Are Crucial for the Binding of Lipoprotein Lipase *
Article Snippet: 24 h after the electroporation, the cells were incubated for 2 h at 4 °C with V5-tagged human LPL (400 μl/well). .. At the end of the incubation period, the cells were washed six times in ice-cold PBS containing 1 m m MgCl2 and 1 m m CaCl2 , and cell extracts were collected in radioimmuno precipitation assay buffer (PBS containing 1% Triton X-100, 0.5% deoxycholate, and 0.02% SDS) along with complete mini EDTA-free protease inhibitors (Roche Applied Science).

Article Title: Highly Conserved Cysteines within the Ly6 Domain of GPIHBP1 Are Crucial for the Binding of Lipoprotein Lipase *
Article Snippet: 24 h after the electroporation, the cells were incubated with PIPLC (5 units/ml) in serum-free medium for 1 h at 37 °C. .. At the end of the incubation, the medium was harvested, and cell extracts were collected in radioimmuno precipitation assay buffer containing complete mini EDTA-free protease inhibitors (Roche Applied Science).

Article Title: Highly Conserved Cysteines within the Ly6 Domain of GPIHBP1 Are Crucial for the Binding of Lipoprotein Lipase *
Article Snippet: 24 h after the electroporation, the cells were incubated with a goat polyclonal antibody against the S-protein tag (1:400 for 2 h at 4 °C) in ice-cold PBS containing 1 m m MgCl2 and 1 m m CaCl2 (PBS/Mg/Ca). .. At the end of the incubation period, the cells were washed six times in ice-cold PBS/Mg/Ca, and the cell extracts were collected in radioimmuno precipitation assay buffer containing complete mini EDTA-free protease inhibitors (Roche Applied Science).

Transfection:

Article Title: Highly Conserved Cysteines within the Ly6 Domain of GPIHBP1 Are Crucial for the Binding of Lipoprotein Lipase *
Article Snippet: Paragraph title: Assessing Amounts of GPIHBP1 Proteins at the Surface of Transfected Cells ... At the end of the incubation, the medium was harvested, and cell extracts were collected in radioimmuno precipitation assay buffer containing complete mini EDTA-free protease inhibitors (Roche Applied Science).

Protease Inhibitor:

Article Title: Epigenetics of programmed obesity: alteration in IUGR rat hepatic IGF1 mRNA expression and histone structure in rapid vs. delayed postnatal catch-up growth
Article Snippet: .. Protein was extracted from day 1 and adult liver using a radioimmuno precipitation assay buffer that contained a protease inhibitor cocktail (Roche, Manheim, Germany). .. Supernatant protein concentration was determined by BCA assay (Pierce, Rockford, IL).

Article Title: TGF-? receptor II in epithelia versus mesenchyme plays distinct roles in the developing lung
Article Snippet: .. Briefly, fresh lung tissues were lysed on ice in radioimmuno precipitation assay buffer containing 1 mM phenyl-methyl sulphonyl fluoride, protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland) and 1 mM sodium orthovanadate. ..

Article Title: Cyclical stretch induces structural changes in atrial myocytes
Article Snippet: .. Western blotting Proteins from whole cell lysates were isolated using radioimmuno-precipitation assay buffer [0.5% sodium deoxycholate, 0.1% sodium dodecylsulfate (SDS) and 1% Igepal ca-630 in TBS] supplemented with a protease inhibitor cocktail (Roche Diagnostics Corp., Indianapolis, IN, USA), a phosphatase inhibitor cocktail, PMSF (1 mmol/l; Roche Diagnostics Corp.) and sodium vanadate (15 mmol/l). .. SDS sample buffer was added and samples were denaturized by heat at 99°C for 5 min. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel, transferred to PVDF membranes (Bio-Rad) and blocked with 5% milk in 0.1% tween in TBS (TBST).

Article Title: Angiotensin type 2 receptor activation promotes browning of white adipose tissue and brown adipogenesis
Article Snippet: .. After measuring the weights of body and fat tissues (inguinal WAT, IgWAT; epididymal WAT, EpiWAT; interscapular BAT), IgWAT were removed and homogenized in radioimmuno-precipitation assay buffer (containing protease inhibitor mixture, Roche Applied Science, Indianapolis, IN, USA) for further immunoblot analyses. ..

Article Title: Glycosylphosphatidyl Inositol-anchored Proteins and fyn Kinase Assemble in Noncaveolar Plasma Membrane Microdomains Defined by Reggie-1 and -2
Article Snippet: .. Two methods were used: 1) Rat brain tissue, DRGs, and pelleted cells were homogenized on ice in modified radioimmuno precipitation-assay buffer ( ) containing 1% NP-40 (Roche Molecular Biochemicals), 0.5% sodium deoxycholate (Sigma), and 0.2% SDS (Sigma) as well as a protease inhibitor cocktail (Complete Mini; Roche Molecular Biochemicals, Mannheim, Germany). .. The homogenates were incubated on ice and insoluble fractions removed by centrifugation.

Radio IP Assay:

Article Title: Oncogenic Ras/Src cooperativity in pancreatic neoplasia
Article Snippet: .. Both tissues and tumor cell lines were lysed in a modified radioimmuno precipitation assay buffer containing 50 m m Tris pH 7.5 at 4°C, 150 m m NaCl, 1 m m EDTA, 50 m m NaF, 5 m m sodium pyrophosphate, 10 m m β-glycerophosphate, 1% NP-40, 1 m m Na3VO4, 0.25% sodium deoxycholate, 0.1% SDS, phosphatase and protease inhibitors (Roche, Indianapolis, IN, USA). .. Lysates were cleared by centrifugation and the protein concentration of the cleared lysate was determined by the bicinchoninic acid method.

Article Title: Epigenetics of programmed obesity: alteration in IUGR rat hepatic IGF1 mRNA expression and histone structure in rapid vs. delayed postnatal catch-up growth
Article Snippet: .. Protein was extracted from day 1 and adult liver using a radioimmuno precipitation assay buffer that contained a protease inhibitor cocktail (Roche, Manheim, Germany). .. Supernatant protein concentration was determined by BCA assay (Pierce, Rockford, IL).

Article Title: Highly Conserved Cysteines within the Ly6 Domain of GPIHBP1 Are Crucial for the Binding of Lipoprotein Lipase *
Article Snippet: .. At the end of the incubation period, the cells were washed six times in ice-cold PBS containing 1 m m MgCl2 and 1 m m CaCl2 , and cell extracts were collected in radioimmuno precipitation assay buffer (PBS containing 1% Triton X-100, 0.5% deoxycholate, and 0.02% SDS) along with complete mini EDTA-free protease inhibitors (Roche Applied Science). .. The amounts of LPL and GPIHBP1 in cell extracts were assessed by Western blotting.

Article Title: Highly Conserved Cysteines within the Ly6 Domain of GPIHBP1 Are Crucial for the Binding of Lipoprotein Lipase *
Article Snippet: .. At the end of the incubation, the medium was harvested, and cell extracts were collected in radioimmuno precipitation assay buffer containing complete mini EDTA-free protease inhibitors (Roche Applied Science). .. In other experiments, we assessed the amount of GPIHBP1 expression on the surface of cells compared with the total amount of GPIHBP1 within cells.

Article Title: Loss of giant obscurins from breast epithelium promotes epithelial-to-mesenchymal transition, tumorigenicity and metastasis
Article Snippet: .. Tissue homogenates were prepared on ice with a hand-homogenizer in radioimmuno-precipitation assay buffer supplemented with a cocktail of protease inhibitors (Roche, Mannheim, Germany) and phosphatase inhibitors (200 nM Imidazole, 100mM Sodium Flouride, 115 mM Sodium Molybdate, 100 mM Sodium Orthovanadate, 400 mM Sodium Tartrate Dihydrate, 100 mM β-Glycerophosphate, 100 mM Sodium Pyrophosphate and 10 mM EGTA), as previously described. .. Protein lysates were electrophoresed on sodium dodecyl sulfate-NuPAGE gel (Invitrogen), transferred to nitrocellulose membranes and probed with primary antibodies as specified in the text.

Article Title: Highly Conserved Cysteines within the Ly6 Domain of GPIHBP1 Are Crucial for the Binding of Lipoprotein Lipase *
Article Snippet: .. At the end of the incubation period, the cells were washed six times in ice-cold PBS/Mg/Ca, and the cell extracts were collected in radioimmuno precipitation assay buffer containing complete mini EDTA-free protease inhibitors (Roche Applied Science). .. To judge the amount of GPIHBP1 on the cell surface, we performed Western blots on cell extracts with an IRdye680-conjugated donkey anti-goat IgG (Li-Cor; 1:800).

Article Title: Adapted HCV JFH1 variant is capable of accommodating a large foreign gene insert and allows lower level HCV replication and viral production
Article Snippet: .. The HCV-transfected Huh 7.5 cells were lysed in a radioimmuno-precipitation assay buffer (50 mM Tris-HCl, pH 7.5, 150 mM sodium chloride, 1% Nonidet P40, 0.5% sodium deoxycholate) containing a cocktail of proteinase inhibitors (Roche). ..

Article Title: Optimisation of Intestinal Fibrosis and Survival in the Mouse S. Typhimurium Model for Anti-fibrotic Drug Discovery and Preclinical Applications
Article Snippet: .. Whole tissue was pulverized under liquid nitrogen and lysed in ice-cold radioimmuno precipitation assay buffer [RIPA buffer] with a cocktail of proteinase inhibitors [Roche, Indianapolis, IN]., Protein content was determined using a modified Bradford assay kit [BioRad, Hercules, CA]. .. Total protein was separated by sodium dodecyl sulfate [SDS] polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride [PVDF] membranes [Amersham Biosciences, Piscataway, NJ].

Article Title: TGF-? receptor II in epithelia versus mesenchyme plays distinct roles in the developing lung
Article Snippet: .. Briefly, fresh lung tissues were lysed on ice in radioimmuno precipitation assay buffer containing 1 mM phenyl-methyl sulphonyl fluoride, protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland) and 1 mM sodium orthovanadate. ..

Article Title: Adapted HCV JFH1 variant is capable of accommodating a large foreign gene insert and allows lower level HCV replication and viral production
Article Snippet: .. Western blot analysis The HCV-transfected Huh 7.5 cells were lysed in a radioimmuno-precipitation assay buffer (50 mM Tris-HCl, pH 7.5, 150 mM sodium chloride, 1% Nonidet P40, 0.5% sodium deoxycholate) containing a cocktail of proteinase inhibitors (Roche). ..

Article Title: Cyclical stretch induces structural changes in atrial myocytes
Article Snippet: .. Western blotting Proteins from whole cell lysates were isolated using radioimmuno-precipitation assay buffer [0.5% sodium deoxycholate, 0.1% sodium dodecylsulfate (SDS) and 1% Igepal ca-630 in TBS] supplemented with a protease inhibitor cocktail (Roche Diagnostics Corp., Indianapolis, IN, USA), a phosphatase inhibitor cocktail, PMSF (1 mmol/l; Roche Diagnostics Corp.) and sodium vanadate (15 mmol/l). .. SDS sample buffer was added and samples were denaturized by heat at 99°C for 5 min. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel, transferred to PVDF membranes (Bio-Rad) and blocked with 5% milk in 0.1% tween in TBS (TBST).

Article Title: Angiotensin type 2 receptor activation promotes browning of white adipose tissue and brown adipogenesis
Article Snippet: .. After measuring the weights of body and fat tissues (inguinal WAT, IgWAT; epididymal WAT, EpiWAT; interscapular BAT), IgWAT were removed and homogenized in radioimmuno-precipitation assay buffer (containing protease inhibitor mixture, Roche Applied Science, Indianapolis, IN, USA) for further immunoblot analyses. ..

Article Title: Glycosylphosphatidyl Inositol-anchored Proteins and fyn Kinase Assemble in Noncaveolar Plasma Membrane Microdomains Defined by Reggie-1 and -2
Article Snippet: .. Two methods were used: 1) Rat brain tissue, DRGs, and pelleted cells were homogenized on ice in modified radioimmuno precipitation-assay buffer ( ) containing 1% NP-40 (Roche Molecular Biochemicals), 0.5% sodium deoxycholate (Sigma), and 0.2% SDS (Sigma) as well as a protease inhibitor cocktail (Complete Mini; Roche Molecular Biochemicals, Mannheim, Germany). .. The homogenates were incubated on ice and insoluble fractions removed by centrifugation.

Imaging:

Article Title: Angiotensin type 2 receptor activation promotes browning of white adipose tissue and brown adipogenesis
Article Snippet: Based on the previous studies, mice were subjected to intraperitoneal injection of ZD7155 (1 mg kg−1 day) or M024/C21 (0.1 mg kg−1 per day) dissolved in 100 μl of phosphate buffer saline (PBS) for 14 days., Body surface temperatures (which correlates to body core temperature and oxygen consumption rate) were monitored at room temperature (RT) by taking the thermographic images of mice using infrared thermal imaging camera (Flir T420; the thermal sensitivity, < 0.045 °C and the spectral range, 7.5–13 μm) and analyzing with a FLIR-Tools software (FLIR System Inc, Wilsonville, OR, USA). .. After measuring the weights of body and fat tissues (inguinal WAT, IgWAT; epididymal WAT, EpiWAT; interscapular BAT), IgWAT were removed and homogenized in radioimmuno-precipitation assay buffer (containing protease inhibitor mixture, Roche Applied Science, Indianapolis, IN, USA) for further immunoblot analyses.

Protein Concentration:

Article Title: Oncogenic Ras/Src cooperativity in pancreatic neoplasia
Article Snippet: Both tissues and tumor cell lines were lysed in a modified radioimmuno precipitation assay buffer containing 50 m m Tris pH 7.5 at 4°C, 150 m m NaCl, 1 m m EDTA, 50 m m NaF, 5 m m sodium pyrophosphate, 10 m m β-glycerophosphate, 1% NP-40, 1 m m Na3VO4, 0.25% sodium deoxycholate, 0.1% SDS, phosphatase and protease inhibitors (Roche, Indianapolis, IN, USA). .. Lysates were cleared by centrifugation and the protein concentration of the cleared lysate was determined by the bicinchoninic acid method.

Article Title: Epigenetics of programmed obesity: alteration in IUGR rat hepatic IGF1 mRNA expression and histone structure in rapid vs. delayed postnatal catch-up growth
Article Snippet: Protein was extracted from day 1 and adult liver using a radioimmuno precipitation assay buffer that contained a protease inhibitor cocktail (Roche, Manheim, Germany). .. Protein was extracted from day 1 and adult liver using a radioimmuno precipitation assay buffer that contained a protease inhibitor cocktail (Roche, Manheim, Germany).

Injection:

Article Title: Angiotensin type 2 receptor activation promotes browning of white adipose tissue and brown adipogenesis
Article Snippet: Based on the previous studies, mice were subjected to intraperitoneal injection of ZD7155 (1 mg kg−1 day) or M024/C21 (0.1 mg kg−1 per day) dissolved in 100 μl of phosphate buffer saline (PBS) for 14 days., Body surface temperatures (which correlates to body core temperature and oxygen consumption rate) were monitored at room temperature (RT) by taking the thermographic images of mice using infrared thermal imaging camera (Flir T420; the thermal sensitivity, < 0.045 °C and the spectral range, 7.5–13 μm) and analyzing with a FLIR-Tools software (FLIR System Inc, Wilsonville, OR, USA). .. After measuring the weights of body and fat tissues (inguinal WAT, IgWAT; epididymal WAT, EpiWAT; interscapular BAT), IgWAT were removed and homogenized in radioimmuno-precipitation assay buffer (containing protease inhibitor mixture, Roche Applied Science, Indianapolis, IN, USA) for further immunoblot analyses.

Binding Assay:

Article Title: Highly Conserved Cysteines within the Ly6 Domain of GPIHBP1 Are Crucial for the Binding of Lipoprotein Lipase *
Article Snippet: Paragraph title: Binding of Human LPL to GPIHBP1-expressing CHO cells ... At the end of the incubation period, the cells were washed six times in ice-cold PBS containing 1 m m MgCl2 and 1 m m CaCl2 , and cell extracts were collected in radioimmuno precipitation assay buffer (PBS containing 1% Triton X-100, 0.5% deoxycholate, and 0.02% SDS) along with complete mini EDTA-free protease inhibitors (Roche Applied Science).

Article Title: Highly Conserved Cysteines within the Ly6 Domain of GPIHBP1 Are Crucial for the Binding of Lipoprotein Lipase *
Article Snippet: At the end of the incubation period, the cells were washed six times in ice-cold PBS/Mg/Ca, and the cell extracts were collected in radioimmuno precipitation assay buffer containing complete mini EDTA-free protease inhibitors (Roche Applied Science). .. The binding of antibodies was detected with an IRdye800-conjugated donkey anti-rat IgG (Li-Cor, 1:2000) or donkey anti-mouse IgG (Li-Cor; 1:2000).

Isolation:

Article Title: Cyclical stretch induces structural changes in atrial myocytes
Article Snippet: .. Western blotting Proteins from whole cell lysates were isolated using radioimmuno-precipitation assay buffer [0.5% sodium deoxycholate, 0.1% sodium dodecylsulfate (SDS) and 1% Igepal ca-630 in TBS] supplemented with a protease inhibitor cocktail (Roche Diagnostics Corp., Indianapolis, IN, USA), a phosphatase inhibitor cocktail, PMSF (1 mmol/l; Roche Diagnostics Corp.) and sodium vanadate (15 mmol/l). .. SDS sample buffer was added and samples were denaturized by heat at 99°C for 5 min. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel, transferred to PVDF membranes (Bio-Rad) and blocked with 5% milk in 0.1% tween in TBS (TBST).

Microscopy:

Article Title: Angiotensin type 2 receptor activation promotes browning of white adipose tissue and brown adipogenesis
Article Snippet: After measuring the weights of body and fat tissues (inguinal WAT, IgWAT; epididymal WAT, EpiWAT; interscapular BAT), IgWAT were removed and homogenized in radioimmuno-precipitation assay buffer (containing protease inhibitor mixture, Roche Applied Science, Indianapolis, IN, USA) for further immunoblot analyses. .. After embedding in FSC22 Frozen Section Media (Leica Microsystem, Buffalo Grove, IL, USA), cryosections (10–20 μm thick) were prepared using a cryostat (CM1950, Leica Microsystems), and images were taken using an Olympus microscope equipped with a digital camera.

Mouse Assay:

Article Title: Angiotensin type 2 receptor activation promotes browning of white adipose tissue and brown adipogenesis
Article Snippet: The mice sera were also collected for further analyses, before mice were killed by a lethal dose of carbondioxide. .. After measuring the weights of body and fat tissues (inguinal WAT, IgWAT; epididymal WAT, EpiWAT; interscapular BAT), IgWAT were removed and homogenized in radioimmuno-precipitation assay buffer (containing protease inhibitor mixture, Roche Applied Science, Indianapolis, IN, USA) for further immunoblot analyses.

Polyacrylamide Gel Electrophoresis:

Article Title: Optimisation of Intestinal Fibrosis and Survival in the Mouse S. Typhimurium Model for Anti-fibrotic Drug Discovery and Preclinical Applications
Article Snippet: Whole tissue was pulverized under liquid nitrogen and lysed in ice-cold radioimmuno precipitation assay buffer [RIPA buffer] with a cocktail of proteinase inhibitors [Roche, Indianapolis, IN]., Protein content was determined using a modified Bradford assay kit [BioRad, Hercules, CA]. .. Total protein was separated by sodium dodecyl sulfate [SDS] polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride [PVDF] membranes [Amersham Biosciences, Piscataway, NJ].

SDS Page:

Article Title: TGF-? receptor II in epithelia versus mesenchyme plays distinct roles in the developing lung
Article Snippet: Briefly, fresh lung tissues were lysed on ice in radioimmuno precipitation assay buffer containing 1 mM phenyl-methyl sulphonyl fluoride, protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland) and 1 mM sodium orthovanadate. .. Equal amounts (40 μg) of total tissue lysate proteins were separated in NuPAGE® 4–12% gradient SDS-PAGE gels using a MOPS buffering system (Invitrogen, Carlsbad, CA, USA).

Plasmid Preparation:

Article Title: Highly Conserved Cysteines within the Ly6 Domain of GPIHBP1 Are Crucial for the Binding of Lipoprotein Lipase *
Article Snippet: 5 × 106 CHO pgsA-745 cells were electroporated with 5 μg of plasmid DNA and seeded in triplicate wells of a 24-well tissue culture plate. .. At the end of the incubation period, the cells were washed six times in ice-cold PBS containing 1 m m MgCl2 and 1 m m CaCl2 , and cell extracts were collected in radioimmuno precipitation assay buffer (PBS containing 1% Triton X-100, 0.5% deoxycholate, and 0.02% SDS) along with complete mini EDTA-free protease inhibitors (Roche Applied Science).

Article Title: Highly Conserved Cysteines within the Ly6 Domain of GPIHBP1 Are Crucial for the Binding of Lipoprotein Lipase *
Article Snippet: CHO pgsA-745 cells (1 × 106 cells) were electroporated with 2 μg of plasmid DNA and seeded in duplicate wells of a 24-well plate. .. At the end of the incubation, the medium was harvested, and cell extracts were collected in radioimmuno precipitation assay buffer containing complete mini EDTA-free protease inhibitors (Roche Applied Science).

Article Title: Highly Conserved Cysteines within the Ly6 Domain of GPIHBP1 Are Crucial for the Binding of Lipoprotein Lipase *
Article Snippet: CHO pgsA-745 cells (2 × 106 cells) were electroporated with 4 μg of plasmid and then seeded in four wells of a 24-well plate. .. At the end of the incubation period, the cells were washed six times in ice-cold PBS/Mg/Ca, and the cell extracts were collected in radioimmuno precipitation assay buffer containing complete mini EDTA-free protease inhibitors (Roche Applied Science).

Software:

Article Title: Angiotensin type 2 receptor activation promotes browning of white adipose tissue and brown adipogenesis
Article Snippet: Based on the previous studies, mice were subjected to intraperitoneal injection of ZD7155 (1 mg kg−1 day) or M024/C21 (0.1 mg kg−1 per day) dissolved in 100 μl of phosphate buffer saline (PBS) for 14 days., Body surface temperatures (which correlates to body core temperature and oxygen consumption rate) were monitored at room temperature (RT) by taking the thermographic images of mice using infrared thermal imaging camera (Flir T420; the thermal sensitivity, < 0.045 °C and the spectral range, 7.5–13 μm) and analyzing with a FLIR-Tools software (FLIR System Inc, Wilsonville, OR, USA). .. After measuring the weights of body and fat tissues (inguinal WAT, IgWAT; epididymal WAT, EpiWAT; interscapular BAT), IgWAT were removed and homogenized in radioimmuno-precipitation assay buffer (containing protease inhibitor mixture, Roche Applied Science, Indianapolis, IN, USA) for further immunoblot analyses.

Immunoprecipitation:

Article Title: Oncogenic Ras/Src cooperativity in pancreatic neoplasia
Article Snippet: Paragraph title: Immunoprecipitation and immunoblot analysis ... Both tissues and tumor cell lines were lysed in a modified radioimmuno precipitation assay buffer containing 50 m m Tris pH 7.5 at 4°C, 150 m m NaCl, 1 m m EDTA, 50 m m NaF, 5 m m sodium pyrophosphate, 10 m m β-glycerophosphate, 1% NP-40, 1 m m Na3VO4, 0.25% sodium deoxycholate, 0.1% SDS, phosphatase and protease inhibitors (Roche, Indianapolis, IN, USA).

Article Title: Glycosylphosphatidyl Inositol-anchored Proteins and fyn Kinase Assemble in Noncaveolar Plasma Membrane Microdomains Defined by Reggie-1 and -2
Article Snippet: Paragraph title: Immunoprecipitation Experiments ... Two methods were used: 1) Rat brain tissue, DRGs, and pelleted cells were homogenized on ice in modified radioimmuno precipitation-assay buffer ( ) containing 1% NP-40 (Roche Molecular Biochemicals), 0.5% sodium deoxycholate (Sigma), and 0.2% SDS (Sigma) as well as a protease inhibitor cocktail (Complete Mini; Roche Molecular Biochemicals, Mannheim, Germany).

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  • 99
    Roche radioimmune precipitation buffer
    ERK is involved in the NF- κ B binding to DNA A , Hs294T melanoma cells were transfected with either empty vector or ERK dominant negative or dominant active constructs. Twenty hours after transfection, cells were placed in serum-free medium. Forty-eight hours after transfection, cells were harvested, and nuclear extracts were made. 10 μ g of nuclear extracts were incubated with 32 P-labeled NF- κ B or Sp1 oligonucleotide probe in the presence of 1–2 μ g of poly(dI-dC). The reaction mixture was electrophoresed on a 6% native gel, which was dried and processed for autoradiography. The arrow indicates the specific complexes associated with the NF- κ B and the Sp1 probe. The EMSA shown is representative one of three different experiments. Results were quantitatively similar in all three experiments. B , Hs294T cells were incubated with PD98059 (50 μ M) for the indicated time periods, nuclei were extracted, and an NF- κ B EMSA was performed as described for A. C , Hs294T melanoma cells were preincubated with proteosome inhibitor MG115 and cycloheximide for 2 h and then treated with PD98059 (50 μ M) for 10, 30, and 60 min, respectively. The cells were then lysed using <t>radioimmune</t> precipitation buffer, and protein was estimated. 50 μ g of protein were loaded in each lane, and SDS-PAGE analysis under reducing conditions was performed. The effect of PD98059 on phosphorylation of I κ B was determined using phosphospecific I κβ antibody.
    Radioimmune Precipitation Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/radioimmune precipitation buffer/product/Roche
    Average 99 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    radioimmune precipitation buffer - by Bioz Stars, 2020-03
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    83
    Roche radioimmune precipitation assay mripa buffer
    ERK is involved in the NF- κ B binding to DNA A , Hs294T melanoma cells were transfected with either empty vector or ERK dominant negative or dominant active constructs. Twenty hours after transfection, cells were placed in serum-free medium. Forty-eight hours after transfection, cells were harvested, and nuclear extracts were made. 10 μ g of nuclear extracts were incubated with 32 P-labeled NF- κ B or Sp1 oligonucleotide probe in the presence of 1–2 μ g of poly(dI-dC). The reaction mixture was electrophoresed on a 6% native gel, which was dried and processed for autoradiography. The arrow indicates the specific complexes associated with the NF- κ B and the Sp1 probe. The EMSA shown is representative one of three different experiments. Results were quantitatively similar in all three experiments. B , Hs294T cells were incubated with PD98059 (50 μ M) for the indicated time periods, nuclei were extracted, and an NF- κ B EMSA was performed as described for A. C , Hs294T melanoma cells were preincubated with proteosome inhibitor MG115 and cycloheximide for 2 h and then treated with PD98059 (50 μ M) for 10, 30, and 60 min, respectively. The cells were then lysed using <t>radioimmune</t> precipitation buffer, and protein was estimated. 50 μ g of protein were loaded in each lane, and SDS-PAGE analysis under reducing conditions was performed. The effect of PD98059 on phosphorylation of I κ B was determined using phosphospecific I κβ antibody.
    Radioimmune Precipitation Assay Mripa Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 83/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/radioimmune precipitation assay mripa buffer/product/Roche
    Average 83 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    radioimmune precipitation assay mripa buffer - by Bioz Stars, 2020-03
    83/100 stars
      Buy from Supplier

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    ERK is involved in the NF- κ B binding to DNA A , Hs294T melanoma cells were transfected with either empty vector or ERK dominant negative or dominant active constructs. Twenty hours after transfection, cells were placed in serum-free medium. Forty-eight hours after transfection, cells were harvested, and nuclear extracts were made. 10 μ g of nuclear extracts were incubated with 32 P-labeled NF- κ B or Sp1 oligonucleotide probe in the presence of 1–2 μ g of poly(dI-dC). The reaction mixture was electrophoresed on a 6% native gel, which was dried and processed for autoradiography. The arrow indicates the specific complexes associated with the NF- κ B and the Sp1 probe. The EMSA shown is representative one of three different experiments. Results were quantitatively similar in all three experiments. B , Hs294T cells were incubated with PD98059 (50 μ M) for the indicated time periods, nuclei were extracted, and an NF- κ B EMSA was performed as described for A. C , Hs294T melanoma cells were preincubated with proteosome inhibitor MG115 and cycloheximide for 2 h and then treated with PD98059 (50 μ M) for 10, 30, and 60 min, respectively. The cells were then lysed using radioimmune precipitation buffer, and protein was estimated. 50 μ g of protein were loaded in each lane, and SDS-PAGE analysis under reducing conditions was performed. The effect of PD98059 on phosphorylation of I κ B was determined using phosphospecific I κβ antibody.

    Journal: The Journal of biological chemistry

    Article Title: A Novel NF-κB-inducing Kinase-MAPK Signaling Pathway Up-regulates NF-κB Activity in Melanoma Cells *

    doi: 10.1074/jbc.M112210200

    Figure Lengend Snippet: ERK is involved in the NF- κ B binding to DNA A , Hs294T melanoma cells were transfected with either empty vector or ERK dominant negative or dominant active constructs. Twenty hours after transfection, cells were placed in serum-free medium. Forty-eight hours after transfection, cells were harvested, and nuclear extracts were made. 10 μ g of nuclear extracts were incubated with 32 P-labeled NF- κ B or Sp1 oligonucleotide probe in the presence of 1–2 μ g of poly(dI-dC). The reaction mixture was electrophoresed on a 6% native gel, which was dried and processed for autoradiography. The arrow indicates the specific complexes associated with the NF- κ B and the Sp1 probe. The EMSA shown is representative one of three different experiments. Results were quantitatively similar in all three experiments. B , Hs294T cells were incubated with PD98059 (50 μ M) for the indicated time periods, nuclei were extracted, and an NF- κ B EMSA was performed as described for A. C , Hs294T melanoma cells were preincubated with proteosome inhibitor MG115 and cycloheximide for 2 h and then treated with PD98059 (50 μ M) for 10, 30, and 60 min, respectively. The cells were then lysed using radioimmune precipitation buffer, and protein was estimated. 50 μ g of protein were loaded in each lane, and SDS-PAGE analysis under reducing conditions was performed. The effect of PD98059 on phosphorylation of I κ B was determined using phosphospecific I κβ antibody.

    Article Snippet: Whole cell extracts were obtained according to our standard protocol using radioimmune precipitation buffer (phosphate-buffered saline, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS) with complete protease inhibitors (Roche Molecular Biochemicals), phosphatase inhibitors (1 mM sodium orthovanadate, 50 mM sodium fluoride), and 100 μ g/ml phenylmethylsulfonyl fluoride.

    Techniques: Binding Assay, Transfection, Plasmid Preparation, Dominant Negative Mutation, Construct, Incubation, Labeling, Autoradiography, SDS Page

    ASK1 expression is reduced in the presence of overexpressed CHIP. COS-7 cells were transiently transfected with HA-ASK1 with or without CHIP (wild type and H260Q). Twenty-four hours after transfection, cells were lysed in radioimmune precipitation

    Journal: Cell Stress & Chaperones

    Article Title: C-terminus of heat shock protein 70- interacting protein facilitates degradation of apoptosis signal-regulating kinase 1 and inhibits apoptosis signal-regulating kinase 1- dependent apoptosis

    doi: 10.1379/CSC-90R.1

    Figure Lengend Snippet: ASK1 expression is reduced in the presence of overexpressed CHIP. COS-7 cells were transiently transfected with HA-ASK1 with or without CHIP (wild type and H260Q). Twenty-four hours after transfection, cells were lysed in radioimmune precipitation

    Article Snippet: Transfected cells were harvested 24 hours after transfection and were lysed with radioimmune precipitation buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM ethylene glycol tetra acetic acid (EGTA), 0.25% sodium deoxycholate, 1% Nonidet P-40, I mM Na3 VO4 , 50 mM NaF) supplemented with protease inhibitor cocktail (Roche Molecular Biochemicals) and 1 mM phenylmethylsulfonyl fluoride (PMSF).

    Techniques: Expressing, Chromatin Immunoprecipitation, Transfection

    Effects of Rab33B inactivation on HBV transcription, HBV protein expression, and solubility. ( A ) For HBV gene expression profiling, HuH-7 cells were treated with control small interfering RNA (siCon) or the Rab33B-specific small interfering (siRab33B) pool and were retransfected with the pHBV∆HP replicon. In parallel, cells were cotransfected with pHBV∆HP and a GFP plasmid (Con) or the GFP-tagged Rab33B.dn mutant. For qRT-PCR, total mRNAs were isolated, reverse transcribed, and applied to PCR reactions using HBV-specific primer sets. Error bars indicate the standard deviations from the mean of two experiments measured in duplicates. ( B ) Rab33B knockdown reduces detergent-soluble and detergent-insoluble core fractions without affecting L protein levels. HuH-7 cells treated with siCon- or siRab33B-specific duplexes were retransfected with pHBV. Seventy-two h after DNA transfection, cell lysates were prepared with radioimmune precipitation (RIPA) buffer, separated into detergent-soluble (soluble (S)) and -insoluble fractions (pellet (P)), and analyzed by WB for the presence of L and core proteins and endogenous β-actin. The levels of core proteins were quantified by densitometric analysis of Western blots and were demonstrated in percent amount relative to the control cells ( n = 4, mean ± SD).

    Journal: Viruses

    Article Title: Rab33B Controls Hepatitis B Virus Assembly by Regulating Core Membrane Association and Nucleocapsid Processing

    doi: 10.3390/v9060157

    Figure Lengend Snippet: Effects of Rab33B inactivation on HBV transcription, HBV protein expression, and solubility. ( A ) For HBV gene expression profiling, HuH-7 cells were treated with control small interfering RNA (siCon) or the Rab33B-specific small interfering (siRab33B) pool and were retransfected with the pHBV∆HP replicon. In parallel, cells were cotransfected with pHBV∆HP and a GFP plasmid (Con) or the GFP-tagged Rab33B.dn mutant. For qRT-PCR, total mRNAs were isolated, reverse transcribed, and applied to PCR reactions using HBV-specific primer sets. Error bars indicate the standard deviations from the mean of two experiments measured in duplicates. ( B ) Rab33B knockdown reduces detergent-soluble and detergent-insoluble core fractions without affecting L protein levels. HuH-7 cells treated with siCon- or siRab33B-specific duplexes were retransfected with pHBV. Seventy-two h after DNA transfection, cell lysates were prepared with radioimmune precipitation (RIPA) buffer, separated into detergent-soluble (soluble (S)) and -insoluble fractions (pellet (P)), and analyzed by WB for the presence of L and core proteins and endogenous β-actin. The levels of core proteins were quantified by densitometric analysis of Western blots and were demonstrated in percent amount relative to the control cells ( n = 4, mean ± SD).

    Article Snippet: Analysis of Soluble and Insoluble Protein Fractions Transfected HuH-7 cells were lysed for 30 min on ice with radioimmune precipitation buffer (RIPA; 10 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid [EDTA], 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS) supplemented with the protease inhibitor cocktail (Roche).

    Techniques: Expressing, Solubility, Small Interfering RNA, Plasmid Preparation, Mutagenesis, Quantitative RT-PCR, Isolation, Polymerase Chain Reaction, Transfection, Western Blot