Structured Review

Roche ripa lysis buffer
Characterization of <t>anti-apoE</t> antibodies. Four different anti-apoE antibodies were tested for their ability to recognize apoE in Western blotting (A and B), ELISA (C), immunohistochemistry (D–H), and immunoprecipitation (I). (A and B) Cerebral cortical tissues from wild-type and apoe KO mice were lysed with <t>RIPA</t> buffer and equal amounts of total proteins were loaded to each well. In B, the membranes shown in A were overexposed to show a weak apoE band in the membranes probed with HJ6.1, HJ6.2, and HJ6.4 antibody. (C) ELISA plates were coated with anti-apoE antibody (WUE4), and each biotinylated HJ antibody was used as a detection antibody. Optical density at 650 nm was measured with a series of different apoe concentrations. (D–G) Biotinylated HJ antibody was used as a primary antibody in the immunohistochemical staining with APPswe/PS1ΔE9 mice brain tissues. Bar, 50 µm. (H) Biotinylated HJ6.3 antibody was used as a primary antibody in the immunohistochemical staining with brain tissues from apoE-deficient APPswe/PS1ΔE9 mice. Bar, 50 µm. (I and J) Cerebral cortical tissues from wild-type mice were lysed with RIPA buffer, and the RIPA lysates were used for immunoprecipitation with each HJ antibody. ApoE proteins left in the supernatant of postimmunoprecipitation (Post-IP) solution (I) and eluted from protein G–Sepharose beads (J) were detected with a polyclonal anti-apoE antibody (EMD Millipore).
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Images

1) Product Images from "Anti-apoE immunotherapy inhibits amyloid accumulation in a transgenic mouse model of A? amyloidosis"

Article Title: Anti-apoE immunotherapy inhibits amyloid accumulation in a transgenic mouse model of A? amyloidosis

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20121274

Characterization of anti-apoE antibodies. Four different anti-apoE antibodies were tested for their ability to recognize apoE in Western blotting (A and B), ELISA (C), immunohistochemistry (D–H), and immunoprecipitation (I). (A and B) Cerebral cortical tissues from wild-type and apoe KO mice were lysed with RIPA buffer and equal amounts of total proteins were loaded to each well. In B, the membranes shown in A were overexposed to show a weak apoE band in the membranes probed with HJ6.1, HJ6.2, and HJ6.4 antibody. (C) ELISA plates were coated with anti-apoE antibody (WUE4), and each biotinylated HJ antibody was used as a detection antibody. Optical density at 650 nm was measured with a series of different apoe concentrations. (D–G) Biotinylated HJ antibody was used as a primary antibody in the immunohistochemical staining with APPswe/PS1ΔE9 mice brain tissues. Bar, 50 µm. (H) Biotinylated HJ6.3 antibody was used as a primary antibody in the immunohistochemical staining with brain tissues from apoE-deficient APPswe/PS1ΔE9 mice. Bar, 50 µm. (I and J) Cerebral cortical tissues from wild-type mice were lysed with RIPA buffer, and the RIPA lysates were used for immunoprecipitation with each HJ antibody. ApoE proteins left in the supernatant of postimmunoprecipitation (Post-IP) solution (I) and eluted from protein G–Sepharose beads (J) were detected with a polyclonal anti-apoE antibody (EMD Millipore).
Figure Legend Snippet: Characterization of anti-apoE antibodies. Four different anti-apoE antibodies were tested for their ability to recognize apoE in Western blotting (A and B), ELISA (C), immunohistochemistry (D–H), and immunoprecipitation (I). (A and B) Cerebral cortical tissues from wild-type and apoe KO mice were lysed with RIPA buffer and equal amounts of total proteins were loaded to each well. In B, the membranes shown in A were overexposed to show a weak apoE band in the membranes probed with HJ6.1, HJ6.2, and HJ6.4 antibody. (C) ELISA plates were coated with anti-apoE antibody (WUE4), and each biotinylated HJ antibody was used as a detection antibody. Optical density at 650 nm was measured with a series of different apoe concentrations. (D–G) Biotinylated HJ antibody was used as a primary antibody in the immunohistochemical staining with APPswe/PS1ΔE9 mice brain tissues. Bar, 50 µm. (H) Biotinylated HJ6.3 antibody was used as a primary antibody in the immunohistochemical staining with brain tissues from apoE-deficient APPswe/PS1ΔE9 mice. Bar, 50 µm. (I and J) Cerebral cortical tissues from wild-type mice were lysed with RIPA buffer, and the RIPA lysates were used for immunoprecipitation with each HJ antibody. ApoE proteins left in the supernatant of postimmunoprecipitation (Post-IP) solution (I) and eluted from protein G–Sepharose beads (J) were detected with a polyclonal anti-apoE antibody (EMD Millipore).

Techniques Used: Western Blot, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Immunoprecipitation, Mouse Assay, Staining

2) Product Images from "The Nsp12-coding region of type 2 PRRSV is required for viral subgenomic mRNA synthesis"

Article Title: The Nsp12-coding region of type 2 PRRSV is required for viral subgenomic mRNA synthesis

Journal: Emerging Microbes & Infections

doi: 10.1080/22221751.2019.1679010

Nsp12 forms dimers under oxidizing conditions. ( A ) and ( B ) Nsp12 forms dimers, and dimer formation is mediated by covalent disulfide bonds. 2-mercaptoethanol (ß-ME) and dithiothreitol (DTT) block cysteine-mediated dimerization. The N protein was used as a positive control. ( C ) Nsp12 homodimerization due to exposure to the oxidizing extracellular environment after cell lysis. HEK293 T cells were seeded in twelve-well plates and transfected with 2 μg of pCMV-HA-N and ( D ) pCMV-HA-Nsp12 plasmids; at 24 hpt, the cells were treated with RIPA lysis buffer with or without 20 mM N-ethylmaleimide. The lysates were also treated with 4x loading buffer with or without β-ME. ( E ) Nsp12 is not incorporated into virions. MARC-145 cells were infected with PRRSV-HuN4 at an MOI of 0.01; 72 h later, the supernatants were collected and pelleted twice through a 30% sucrose cushion, after which the purified virions were fractionated using 10% to 35% sucrose cushions. Samples were collected from the top to the bottom and analyzed by Western blotting. These experiments were performed three times, and a representative result is shown.
Figure Legend Snippet: Nsp12 forms dimers under oxidizing conditions. ( A ) and ( B ) Nsp12 forms dimers, and dimer formation is mediated by covalent disulfide bonds. 2-mercaptoethanol (ß-ME) and dithiothreitol (DTT) block cysteine-mediated dimerization. The N protein was used as a positive control. ( C ) Nsp12 homodimerization due to exposure to the oxidizing extracellular environment after cell lysis. HEK293 T cells were seeded in twelve-well plates and transfected with 2 μg of pCMV-HA-N and ( D ) pCMV-HA-Nsp12 plasmids; at 24 hpt, the cells were treated with RIPA lysis buffer with or without 20 mM N-ethylmaleimide. The lysates were also treated with 4x loading buffer with or without β-ME. ( E ) Nsp12 is not incorporated into virions. MARC-145 cells were infected with PRRSV-HuN4 at an MOI of 0.01; 72 h later, the supernatants were collected and pelleted twice through a 30% sucrose cushion, after which the purified virions were fractionated using 10% to 35% sucrose cushions. Samples were collected from the top to the bottom and analyzed by Western blotting. These experiments were performed three times, and a representative result is shown.

Techniques Used: Blocking Assay, Positive Control, Lysis, Transfection, Infection, Purification, Western Blot

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Centrifugation:

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Blocking Assay:

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Incubation:

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BIA-KA:

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Modification:

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Western Blot:

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Derivative Assay:

Article Title: Sigma-1 receptor expression in sensory neurons and the effect of painful peripheral nerve injury
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Transfection:

Article Title: The Nsp12-coding region of type 2 PRRSV is required for viral subgenomic mRNA synthesis
Article Snippet: Paragraph title: Transfection and Western blotting ... At 48 h posttransfection (hpt), the cells were collected, washed once with PBS and lysed in RIPA Lysis Buffer containing a protease inhibitor cocktail (Roche).

Concentration Assay:

Article Title: EphA1 activation promotes the homing of endothelial progenitor cells to hepatocellular carcinoma for tumor neovascularization through the SDF-1/CXCR4 signaling pathway
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Protease Inhibitor:

Article Title: The combination of Cassia obtusifolia L. and Foeniculum vulgare M. exhibits a laxative effect on loperamide-induced constipation of rats
Article Snippet: .. Western blot analysis Protein samples were prepared from the transverse colon using RIPA lysis buffer supplemented with protease inhibitor cocktail (Roche Dignostics, Mannheim, Germany) and phosphatase inhibitor cocktail (Roche Dignostics). .. Protein samples were separated on SDS-PAGE gels and transferred to PVDF membranes (Millipore, Billerica, USA).

Article Title: Epigenetic regulation of diacylglycerol kinase alpha promotes radiation-induced fibrosis
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Article Title: EphA1 activation promotes the homing of endothelial progenitor cells to hepatocellular carcinoma for tumor neovascularization through the SDF-1/CXCR4 signaling pathway
Article Snippet: .. SDF-1 and CXCR4 co-immunoprecipitation (IP) EPCs collected from the co-culture experiment were washed twice with ice-cold PBS and lysed via incubation on ice for 30 min in 1 ml of modified RIPA lysis buffer containing 50 mM Tris–HCl, pH 7.4, 1 % NP-40, 0.25 % Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, aprotinin, leupeptin, and pepstatin: 1 μg/ml each,1 mM Na3 VO4 , 1 mM NaF, and a commercial protease inhibitor mixture (Complete-Mini Protease Inhibitor Mixture; Roche Diagnostics). .. After the proteins were quantified and the concentration was adjusted with PBS to approximately 2 μg/μl, the cell lysate was pre-cleared by adding 20 μl of protein G sepharose bead slurry per ml of cell lysate and incubating at 4 °C for 1 h on a rocker.

Article Title: Tenascin-C, a Prognostic Determinant of Esophageal Squamous Cell Carcinoma
Article Snippet: .. Western blotting analysis Cell lysates were produced in RIPA lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton x-100, 1% Na-Doc, 0.1% SDS) supplemented with protease inhibitor cocktail (Roche). .. Cell extracts were quantitated using a BCA protein assay kit (Thermo).

Article Title: The Nsp12-coding region of type 2 PRRSV is required for viral subgenomic mRNA synthesis
Article Snippet: .. At 48 h posttransfection (hpt), the cells were collected, washed once with PBS and lysed in RIPA Lysis Buffer containing a protease inhibitor cocktail (Roche). ..

Article Title: Anti-apoE immunotherapy inhibits amyloid accumulation in a transgenic mouse model of A? amyloidosis
Article Snippet: .. Cerebral cortical tissues from wild-type and apoe KO mice were lysed with RIPA lysis buffer (1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 25 mM Tris-HCl, and 150 mM NaCl) in the presence of 1X protease inhibitor mixture (Roche). ..

Article Title: Energy-requiring uptake of prostasomes and PC3 cell-derived exosomes into non-malignant and malignant cells
Article Snippet: .. Western blot analysis Cells were harvested and homogenized in RIPA lysis buffer (10 mM Tris, pH 7.2, 150 mM NaCl, 1% deoxycholate, 1% TritonX-100, 0.1% SDS, 5 mM EDTA) containing complete protease inhibitor cocktail, phospho-stop (Roche Diagnostics, Meylan, France), DTT (Sigma-Aldrich) and vanadate (Thermo Fisher Scientific). ..

Protein Concentration:

Article Title: Anti-apoE immunotherapy inhibits amyloid accumulation in a transgenic mouse model of A? amyloidosis
Article Snippet: Cerebral cortical tissues from wild-type and apoe KO mice were lysed with RIPA lysis buffer (1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 25 mM Tris-HCl, and 150 mM NaCl) in the presence of 1X protease inhibitor mixture (Roche). .. Protein concentration in supernatants was determined using the BCA protein assay kit (Thermo Fisher Scientific).

Affinity Purification:

Article Title: Sigma-1 receptor expression in sensory neurons and the effect of painful peripheral nerve injury
Article Snippet: Harvested ganglia were homogenized in 150 μl of RIPA lysis buffer containing protease inhibitors (Roche Diagnostics, Indianapolis, IN) and phosphatase inhibitors (Thermo Scientific, Rockford, IL), and incubated on ice for 30 minutes. .. After blocking with 5% milk in TBS-T (Tris-buffered saline plus 0.1% Tween 20), blots were sequentially probed overnight at 4°C with one of two anti-σ1R rabbit polyclonal antibodies, either a commercial product (1:500, Invitrogen, Camarillo, CA, catalog number 42-3300; Lot No. 797152A, polyclonal affinity-purified antibody raised against a synthetic peptide derived from the C-terminus region (amino acid 139-155) of the rat σ1R), or another that was a kind gift from Drs. Su and Hayashi of NIDA (1:1000, Lot No. 6405, polyclonal affinity-purified antibody raised against amino acid 52-69 from the C-terminus of rat σ1R) [ , ].

Isolation:

Article Title: Epigenetic regulation of diacylglycerol kinase alpha promotes radiation-induced fibrosis
Article Snippet: .. Western blot analysis Total protein was isolated by lysing cells in RIPA lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl 1.0% (v/v) nonidet-P40, 0.5% m/v sodium deoxycholate, 0.1% m/v sodium dodecyl sulfate) supplemented with protease inhibitor (Roche). .. Western blot analysis was run with NuPAGE SDS-PAGE gel system (Life Technologies) and primary antibodies against DGKA (11547-1-AP, Proteintech), beta actin (ACTB; sc-47778 HRP, Santa Cruz Biotechnology), PRKCA (2056P, Cell Signaling Technology), collagen 1 (EPR7785, Abcam) and appropriate secondary antibodies (Santa Cruz Biotechnology).

Article Title: Sigma-1 receptor expression in sensory neurons and the effect of painful peripheral nerve injury
Article Snippet: Western blot analysis Total protein was isolated from individual L4 and L5 DRGs of Control animals, and separately from L4 and L5 DRGs of SNL animals. .. Harvested ganglia were homogenized in 150 μl of RIPA lysis buffer containing protease inhibitors (Roche Diagnostics, Indianapolis, IN) and phosphatase inhibitors (Thermo Scientific, Rockford, IL), and incubated on ice for 30 minutes.

Bicinchoninic Acid Protein Assay:

Article Title: Sigma-1 receptor expression in sensory neurons and the effect of painful peripheral nerve injury
Article Snippet: Harvested ganglia were homogenized in 150 μl of RIPA lysis buffer containing protease inhibitors (Roche Diagnostics, Indianapolis, IN) and phosphatase inhibitors (Thermo Scientific, Rockford, IL), and incubated on ice for 30 minutes. .. The supernatant was used for protein estimation using Pierce bicinchoninic acid protein assay kit (Thermo Scientific, Rockford, IL).

Mouse Assay:

Article Title: Anti-apoE immunotherapy inhibits amyloid accumulation in a transgenic mouse model of A? amyloidosis
Article Snippet: .. Cerebral cortical tissues from wild-type and apoe KO mice were lysed with RIPA lysis buffer (1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 25 mM Tris-HCl, and 150 mM NaCl) in the presence of 1X protease inhibitor mixture (Roche). ..

Co-Culture Assay:

Article Title: EphA1 activation promotes the homing of endothelial progenitor cells to hepatocellular carcinoma for tumor neovascularization through the SDF-1/CXCR4 signaling pathway
Article Snippet: .. SDF-1 and CXCR4 co-immunoprecipitation (IP) EPCs collected from the co-culture experiment were washed twice with ice-cold PBS and lysed via incubation on ice for 30 min in 1 ml of modified RIPA lysis buffer containing 50 mM Tris–HCl, pH 7.4, 1 % NP-40, 0.25 % Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, aprotinin, leupeptin, and pepstatin: 1 μg/ml each,1 mM Na3 VO4 , 1 mM NaF, and a commercial protease inhibitor mixture (Complete-Mini Protease Inhibitor Mixture; Roche Diagnostics). .. After the proteins were quantified and the concentration was adjusted with PBS to approximately 2 μg/μl, the cell lysate was pre-cleared by adding 20 μl of protein G sepharose bead slurry per ml of cell lysate and incubating at 4 °C for 1 h on a rocker.

Polyacrylamide Gel Electrophoresis:

Article Title: Sigma-1 receptor expression in sensory neurons and the effect of painful peripheral nerve injury
Article Snippet: Harvested ganglia were homogenized in 150 μl of RIPA lysis buffer containing protease inhibitors (Roche Diagnostics, Indianapolis, IN) and phosphatase inhibitors (Thermo Scientific, Rockford, IL), and incubated on ice for 30 minutes. .. Equal amounts of protein (30 μg) were separated on 4-15% sodium dodecyl sulphate polyacrylamide gel electrophoresis gel (SDS-PAGE; Bio-Rad, Hercules, CA) and transferred onto a polyvinylidene fluoride membrane.

SDS Page:

Article Title: The combination of Cassia obtusifolia L. and Foeniculum vulgare M. exhibits a laxative effect on loperamide-induced constipation of rats
Article Snippet: Western blot analysis Protein samples were prepared from the transverse colon using RIPA lysis buffer supplemented with protease inhibitor cocktail (Roche Dignostics, Mannheim, Germany) and phosphatase inhibitor cocktail (Roche Dignostics). .. Protein samples were separated on SDS-PAGE gels and transferred to PVDF membranes (Millipore, Billerica, USA).

Article Title: Epigenetic regulation of diacylglycerol kinase alpha promotes radiation-induced fibrosis
Article Snippet: Western blot analysis Total protein was isolated by lysing cells in RIPA lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl 1.0% (v/v) nonidet-P40, 0.5% m/v sodium deoxycholate, 0.1% m/v sodium dodecyl sulfate) supplemented with protease inhibitor (Roche). .. Western blot analysis was run with NuPAGE SDS-PAGE gel system (Life Technologies) and primary antibodies against DGKA (11547-1-AP, Proteintech), beta actin (ACTB; sc-47778 HRP, Santa Cruz Biotechnology), PRKCA (2056P, Cell Signaling Technology), collagen 1 (EPR7785, Abcam) and appropriate secondary antibodies (Santa Cruz Biotechnology).

Article Title: Myosin VI-Dependent Actin Cages Encapsulate Parkin-Positive Damaged Mitochondria
Article Snippet: .. Cell lysates were prepared in ice-cold RIPA lysis buffer (1% Triton, 0.1% SDS, 1% DOC, 150 mM NaCl, 10 mM Tris, pH 7.4, 5 mM EDTA) containing protease inhibitors (cOmplete mini, EDTA-free, Roche), boiled in SDS loading buffer, and separated by SDS-PAGE. .. After protein transfer on to PVDF (Immobilon-P, Millipore), the membrane was blocked with 5% fat-free milk powder in PBS-T (0.05% Tween-20 in PBS) for 1 h at room temperature (RT) and incubated with primary antibodies overnight at 4°C.

Article Title: RNF135, RING finger protein, promotes the proliferation of human glioblastoma cells in vivo and in vitro via the ERK pathway
Article Snippet: Western blot analysis Tissues and cell total proteins were collected using RIPA lysis buffer containing phosphatase and protease inhibitors (Roche, Swiss) in accordance with the manufacturer’s instructions. .. Proteins were separated by SDS-PAGE, and then transferred to PVDF membranes (Cat: IPVH00010, EMD Millipore Corporation Billerica Ma01821, USA), blocked with 5% fat-free dry milk or 5% BSA in TBST and immunoblotted with primary antibodies at 4 °C overnight.

Article Title: The Nsp12-coding region of type 2 PRRSV is required for viral subgenomic mRNA synthesis
Article Snippet: At 48 h posttransfection (hpt), the cells were collected, washed once with PBS and lysed in RIPA Lysis Buffer containing a protease inhibitor cocktail (Roche). .. Proteins in the cell lysates were separated by SDS-PAGE, transferred to PVDF membranes (Millipore, Germany) and probed with the indicated antibodies.

Article Title: Sigma-1 receptor expression in sensory neurons and the effect of painful peripheral nerve injury
Article Snippet: Harvested ganglia were homogenized in 150 μl of RIPA lysis buffer containing protease inhibitors (Roche Diagnostics, Indianapolis, IN) and phosphatase inhibitors (Thermo Scientific, Rockford, IL), and incubated on ice for 30 minutes. .. Equal amounts of protein (30 μg) were separated on 4-15% sodium dodecyl sulphate polyacrylamide gel electrophoresis gel (SDS-PAGE; Bio-Rad, Hercules, CA) and transferred onto a polyvinylidene fluoride membrane.

Software:

Article Title: Epigenetic regulation of diacylglycerol kinase alpha promotes radiation-induced fibrosis
Article Snippet: Western blot analysis Total protein was isolated by lysing cells in RIPA lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl 1.0% (v/v) nonidet-P40, 0.5% m/v sodium deoxycholate, 0.1% m/v sodium dodecyl sulfate) supplemented with protease inhibitor (Roche). .. Band intensities were quantitated with ImageJ software using beta actin band intensities for normalization.

Produced:

Article Title: Tenascin-C, a Prognostic Determinant of Esophageal Squamous Cell Carcinoma
Article Snippet: .. Western blotting analysis Cell lysates were produced in RIPA lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton x-100, 1% Na-Doc, 0.1% SDS) supplemented with protease inhibitor cocktail (Roche). .. Cell extracts were quantitated using a BCA protein assay kit (Thermo).

Immunoprecipitation:

Article Title: EphA1 activation promotes the homing of endothelial progenitor cells to hepatocellular carcinoma for tumor neovascularization through the SDF-1/CXCR4 signaling pathway
Article Snippet: SDF-1 and CXCR4 co-immunoprecipitation (IP) EPCs collected from the co-culture experiment were washed twice with ice-cold PBS and lysed via incubation on ice for 30 min in 1 ml of modified RIPA lysis buffer containing 50 mM Tris–HCl, pH 7.4, 1 % NP-40, 0.25 % Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, aprotinin, leupeptin, and pepstatin: 1 μg/ml each,1 mM Na3 VO4 , 1 mM NaF, and a commercial protease inhibitor mixture (Complete-Mini Protease Inhibitor Mixture; Roche Diagnostics). .. SDF-1 and CXCR4 proteins were immunoprecipitated by incubating pre-cleared lysate with rabbit anti-SDF-1 antibody or mouse anti-CXCR4 antibody and protein G-Sepharose (40 μl) overnight at 4 °C.

Lysis:

Article Title: The combination of Cassia obtusifolia L. and Foeniculum vulgare M. exhibits a laxative effect on loperamide-induced constipation of rats
Article Snippet: .. Western blot analysis Protein samples were prepared from the transverse colon using RIPA lysis buffer supplemented with protease inhibitor cocktail (Roche Dignostics, Mannheim, Germany) and phosphatase inhibitor cocktail (Roche Dignostics). .. Protein samples were separated on SDS-PAGE gels and transferred to PVDF membranes (Millipore, Billerica, USA).

Article Title: Epigenetic regulation of diacylglycerol kinase alpha promotes radiation-induced fibrosis
Article Snippet: .. Western blot analysis Total protein was isolated by lysing cells in RIPA lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl 1.0% (v/v) nonidet-P40, 0.5% m/v sodium deoxycholate, 0.1% m/v sodium dodecyl sulfate) supplemented with protease inhibitor (Roche). .. Western blot analysis was run with NuPAGE SDS-PAGE gel system (Life Technologies) and primary antibodies against DGKA (11547-1-AP, Proteintech), beta actin (ACTB; sc-47778 HRP, Santa Cruz Biotechnology), PRKCA (2056P, Cell Signaling Technology), collagen 1 (EPR7785, Abcam) and appropriate secondary antibodies (Santa Cruz Biotechnology).

Article Title: Myosin VI-Dependent Actin Cages Encapsulate Parkin-Positive Damaged Mitochondria
Article Snippet: .. Cell lysates were prepared in ice-cold RIPA lysis buffer (1% Triton, 0.1% SDS, 1% DOC, 150 mM NaCl, 10 mM Tris, pH 7.4, 5 mM EDTA) containing protease inhibitors (cOmplete mini, EDTA-free, Roche), boiled in SDS loading buffer, and separated by SDS-PAGE. .. After protein transfer on to PVDF (Immobilon-P, Millipore), the membrane was blocked with 5% fat-free milk powder in PBS-T (0.05% Tween-20 in PBS) for 1 h at room temperature (RT) and incubated with primary antibodies overnight at 4°C.

Article Title: RNF135, RING finger protein, promotes the proliferation of human glioblastoma cells in vivo and in vitro via the ERK pathway
Article Snippet: .. Western blot analysis Tissues and cell total proteins were collected using RIPA lysis buffer containing phosphatase and protease inhibitors (Roche, Swiss) in accordance with the manufacturer’s instructions. ..

Article Title: EphA1 activation promotes the homing of endothelial progenitor cells to hepatocellular carcinoma for tumor neovascularization through the SDF-1/CXCR4 signaling pathway
Article Snippet: .. SDF-1 and CXCR4 co-immunoprecipitation (IP) EPCs collected from the co-culture experiment were washed twice with ice-cold PBS and lysed via incubation on ice for 30 min in 1 ml of modified RIPA lysis buffer containing 50 mM Tris–HCl, pH 7.4, 1 % NP-40, 0.25 % Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, aprotinin, leupeptin, and pepstatin: 1 μg/ml each,1 mM Na3 VO4 , 1 mM NaF, and a commercial protease inhibitor mixture (Complete-Mini Protease Inhibitor Mixture; Roche Diagnostics). .. After the proteins were quantified and the concentration was adjusted with PBS to approximately 2 μg/μl, the cell lysate was pre-cleared by adding 20 μl of protein G sepharose bead slurry per ml of cell lysate and incubating at 4 °C for 1 h on a rocker.

Article Title: Ursodeoxycholic Acid but Not Tauroursodeoxycholic Acid Inhibits Proliferation and Differentiation of Human Subcutaneous Adipocytes
Article Snippet: .. Western blotting Cells were washed two times with PBS and lysed on ice for 30 minutes in RIPA lysis buffer supplemented with protease and phosphatase inhibitors (Complete, PhoStop, Roche Diagnostics, Mannheim, Germany). .. Protein concentrations were determined using the bicinchoninic assay, Pierce (Rockford, IL, USA).

Article Title: β2-microglobulin is a systemic pro-aging factor that impairs cognitive function and neurogenesis
Article Snippet: .. Western Blot Analysis Mouse hippocampi were dissected after perfusion of animals, snap frozen and lysed in RIPA lysis buffer (500 mM Tris, pH 7.4, 150 mM NaCl, 0.5% Na deoxycholate, 1% NP40, 0.1% SDS, and complete protease inhibitors; Roche). .. Tissue lysates were mixed with 4× NuPage LDS loading buffer (Invitrogen) and loaded on a 4–12% SDS polyacrylamide gradient gel (Invitrogen) and subsequently transferred onto a nitrocellulose membrane.

Article Title: Tenascin-C, a Prognostic Determinant of Esophageal Squamous Cell Carcinoma
Article Snippet: .. Western blotting analysis Cell lysates were produced in RIPA lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton x-100, 1% Na-Doc, 0.1% SDS) supplemented with protease inhibitor cocktail (Roche). .. Cell extracts were quantitated using a BCA protein assay kit (Thermo).

Article Title: The Nsp12-coding region of type 2 PRRSV is required for viral subgenomic mRNA synthesis
Article Snippet: .. At 48 h posttransfection (hpt), the cells were collected, washed once with PBS and lysed in RIPA Lysis Buffer containing a protease inhibitor cocktail (Roche). ..

Article Title: Anti-apoE immunotherapy inhibits amyloid accumulation in a transgenic mouse model of A? amyloidosis
Article Snippet: .. Cerebral cortical tissues from wild-type and apoe KO mice were lysed with RIPA lysis buffer (1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 25 mM Tris-HCl, and 150 mM NaCl) in the presence of 1X protease inhibitor mixture (Roche). ..

Article Title: Energy-requiring uptake of prostasomes and PC3 cell-derived exosomes into non-malignant and malignant cells
Article Snippet: .. Western blot analysis Cells were harvested and homogenized in RIPA lysis buffer (10 mM Tris, pH 7.2, 150 mM NaCl, 1% deoxycholate, 1% TritonX-100, 0.1% SDS, 5 mM EDTA) containing complete protease inhibitor cocktail, phospho-stop (Roche Diagnostics, Meylan, France), DTT (Sigma-Aldrich) and vanadate (Thermo Fisher Scientific). ..

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  • 99
    Roche ripa buffer
    Macrophage migration requires the c-Abl kinase activity. (A–D) <t>BMDM</t> monolayers were wounded with the tip of a pipette and, after washing, added with medium supplemented with 100 ng/ml LPA without (A, B) or plus 10 μM imatinib mesylate (STI) (C) or 10 μM PP2 (D). Monolayers were photographed immediately after the wound (A) or after 6 h from wounding (B–D). (E) Cells migrated into the wound were quantified after different time from wounding. (F) BMDM were incubated with control medium (−) or medium supplemented with 10 μM imatinib mesylate (STI) or 10 μM PP2 for 30 min before lysis. To detect phosphorylation of SFKs, lysates were separated on SDS/PAGE gels, proteins blotted on nitrocellulose and blots probed with Abs of the indicated specificity (WB). To detect c-Abl tyrosine phosphorylation, BMDM were lysed in <t>RIPA</t> buffer as described in Section 2 and immunoprecipitated (IP) with anti-Abl Abs before blotting. (G) BMDM were incubated with control medium (−) or medium supplemented with 10 μM imatinib mesylate (STI) or 10 μM PP2 for 30 min before lysis. (H) Phase contrast images of cells at the margin of the wound after 2 h are shown. These were taken at 40× magnification with an Olympus IX50 microscope and acquired with an Olympus c-7070 wide zoom Digital compact camera. Inhibitors were used at the concentration reported above. One typical of several experiment performed is reported. (I) Baf3 cells expressing Bcr/Abl are strongly polarized. Baf3 cells transfected with an empty vector (Baf3/pSrl) or a vector containing wild-type Bcr/Abl (Baf3/p210wt) or Bcr-Abl with the T315 mutation (Baf3/T315) were cultivated and incubated with STI (10 μM) or PP2 (10 μM) as described in Section 2. Morphology of cells either untreated or treated with STI or PP2 is shown. Cells were plated in 24 well plates in RPMI 1640 medium containing 10% FBS and phase contrast images at 40× magnification taken as described in the legend.
    Ripa Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 4719 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    Roche a498 cells
    Pathogen-specific signaling in infected <t>A498</t> cells.
    A498 Cells, supplied by Roche, used in various techniques. Bioz Stars score: 87/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Roche ripa lysis buffer
    Characterization of <t>anti-apoE</t> antibodies. Four different anti-apoE antibodies were tested for their ability to recognize apoE in Western blotting (A and B), ELISA (C), immunohistochemistry (D–H), and immunoprecipitation (I). (A and B) Cerebral cortical tissues from wild-type and apoe KO mice were lysed with <t>RIPA</t> buffer and equal amounts of total proteins were loaded to each well. In B, the membranes shown in A were overexposed to show a weak apoE band in the membranes probed with HJ6.1, HJ6.2, and HJ6.4 antibody. (C) ELISA plates were coated with anti-apoE antibody (WUE4), and each biotinylated HJ antibody was used as a detection antibody. Optical density at 650 nm was measured with a series of different apoe concentrations. (D–G) Biotinylated HJ antibody was used as a primary antibody in the immunohistochemical staining with APPswe/PS1ΔE9 mice brain tissues. Bar, 50 µm. (H) Biotinylated HJ6.3 antibody was used as a primary antibody in the immunohistochemical staining with brain tissues from apoE-deficient APPswe/PS1ΔE9 mice. Bar, 50 µm. (I and J) Cerebral cortical tissues from wild-type mice were lysed with RIPA buffer, and the RIPA lysates were used for immunoprecipitation with each HJ antibody. ApoE proteins left in the supernatant of postimmunoprecipitation (Post-IP) solution (I) and eluted from protein G–Sepharose beads (J) were detected with a polyclonal anti-apoE antibody (EMD Millipore).
    Ripa Lysis Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 1512 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Macrophage migration requires the c-Abl kinase activity. (A–D) BMDM monolayers were wounded with the tip of a pipette and, after washing, added with medium supplemented with 100 ng/ml LPA without (A, B) or plus 10 μM imatinib mesylate (STI) (C) or 10 μM PP2 (D). Monolayers were photographed immediately after the wound (A) or after 6 h from wounding (B–D). (E) Cells migrated into the wound were quantified after different time from wounding. (F) BMDM were incubated with control medium (−) or medium supplemented with 10 μM imatinib mesylate (STI) or 10 μM PP2 for 30 min before lysis. To detect phosphorylation of SFKs, lysates were separated on SDS/PAGE gels, proteins blotted on nitrocellulose and blots probed with Abs of the indicated specificity (WB). To detect c-Abl tyrosine phosphorylation, BMDM were lysed in RIPA buffer as described in Section 2 and immunoprecipitated (IP) with anti-Abl Abs before blotting. (G) BMDM were incubated with control medium (−) or medium supplemented with 10 μM imatinib mesylate (STI) or 10 μM PP2 for 30 min before lysis. (H) Phase contrast images of cells at the margin of the wound after 2 h are shown. These were taken at 40× magnification with an Olympus IX50 microscope and acquired with an Olympus c-7070 wide zoom Digital compact camera. Inhibitors were used at the concentration reported above. One typical of several experiment performed is reported. (I) Baf3 cells expressing Bcr/Abl are strongly polarized. Baf3 cells transfected with an empty vector (Baf3/pSrl) or a vector containing wild-type Bcr/Abl (Baf3/p210wt) or Bcr-Abl with the T315 mutation (Baf3/T315) were cultivated and incubated with STI (10 μM) or PP2 (10 μM) as described in Section 2. Morphology of cells either untreated or treated with STI or PP2 is shown. Cells were plated in 24 well plates in RPMI 1640 medium containing 10% FBS and phase contrast images at 40× magnification taken as described in the legend.

    Journal: FEBS letters

    Article Title: c-Abl and Src-family kinases cross-talk in regulation of myeloid cell migration

    doi: 10.1016/j.febslet.2009.11.009

    Figure Lengend Snippet: Macrophage migration requires the c-Abl kinase activity. (A–D) BMDM monolayers were wounded with the tip of a pipette and, after washing, added with medium supplemented with 100 ng/ml LPA without (A, B) or plus 10 μM imatinib mesylate (STI) (C) or 10 μM PP2 (D). Monolayers were photographed immediately after the wound (A) or after 6 h from wounding (B–D). (E) Cells migrated into the wound were quantified after different time from wounding. (F) BMDM were incubated with control medium (−) or medium supplemented with 10 μM imatinib mesylate (STI) or 10 μM PP2 for 30 min before lysis. To detect phosphorylation of SFKs, lysates were separated on SDS/PAGE gels, proteins blotted on nitrocellulose and blots probed with Abs of the indicated specificity (WB). To detect c-Abl tyrosine phosphorylation, BMDM were lysed in RIPA buffer as described in Section 2 and immunoprecipitated (IP) with anti-Abl Abs before blotting. (G) BMDM were incubated with control medium (−) or medium supplemented with 10 μM imatinib mesylate (STI) or 10 μM PP2 for 30 min before lysis. (H) Phase contrast images of cells at the margin of the wound after 2 h are shown. These were taken at 40× magnification with an Olympus IX50 microscope and acquired with an Olympus c-7070 wide zoom Digital compact camera. Inhibitors were used at the concentration reported above. One typical of several experiment performed is reported. (I) Baf3 cells expressing Bcr/Abl are strongly polarized. Baf3 cells transfected with an empty vector (Baf3/pSrl) or a vector containing wild-type Bcr/Abl (Baf3/p210wt) or Bcr-Abl with the T315 mutation (Baf3/T315) were cultivated and incubated with STI (10 μM) or PP2 (10 μM) as described in Section 2. Morphology of cells either untreated or treated with STI or PP2 is shown. Cells were plated in 24 well plates in RPMI 1640 medium containing 10% FBS and phase contrast images at 40× magnification taken as described in the legend.

    Article Snippet: BMDM were lysed in RIPA buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 1% (v/v) Triton X-100, 1% Na-deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 5 mM EDTA, 1 mM DTT, 100 μM Na2 VO4 , 10 μM phenilarsine oxide (PAO) and one tablet of Complete™ Mini EDTA-free protease inhibitor cocktail from Roche Molecular Biochemicals for each 10 ml lysis buffer).

    Techniques: Migration, Activity Assay, Transferring, Incubation, Lysis, SDS Page, Western Blot, Immunoprecipitation, Microscopy, Concentration Assay, Expressing, Transfection, Plasmid Preparation, Mutagenesis

    Pathogen-specific signaling in infected A498 cells.

    Journal: The Journal of Clinical Investigation

    Article Title: Bacterial control of host gene expression through RNA polymerase II

    doi: 10.1172/JCI66451

    Figure Lengend Snippet: Pathogen-specific signaling in infected A498 cells.

    Article Snippet: Cell lysates from A498 cells (RIPA lysis buffer supplemented with protease andphosphatase inhibitors; Roche Diagnostics) were separated by SDS-PAGE (4%–12%Bis-Tris gels; Invitrogen), and blotted PVDF membranes were incubated with antibodiesagainst RNA polymerase II subunit B1 (phospho CTD Ser2), clone 3E10 (Millipore), orantibodies against FOSB (Cell Signaling Technology), cFOS-P (Abcam), LBH (Biosite),and DACH1 (Abcam), followed by the appropriate anti–IgG-HRP with ECL PlusWestern Blotting Reagent (GE Healthcare).

    Techniques: Infection

    Characterization of anti-apoE antibodies. Four different anti-apoE antibodies were tested for their ability to recognize apoE in Western blotting (A and B), ELISA (C), immunohistochemistry (D–H), and immunoprecipitation (I). (A and B) Cerebral cortical tissues from wild-type and apoe KO mice were lysed with RIPA buffer and equal amounts of total proteins were loaded to each well. In B, the membranes shown in A were overexposed to show a weak apoE band in the membranes probed with HJ6.1, HJ6.2, and HJ6.4 antibody. (C) ELISA plates were coated with anti-apoE antibody (WUE4), and each biotinylated HJ antibody was used as a detection antibody. Optical density at 650 nm was measured with a series of different apoe concentrations. (D–G) Biotinylated HJ antibody was used as a primary antibody in the immunohistochemical staining with APPswe/PS1ΔE9 mice brain tissues. Bar, 50 µm. (H) Biotinylated HJ6.3 antibody was used as a primary antibody in the immunohistochemical staining with brain tissues from apoE-deficient APPswe/PS1ΔE9 mice. Bar, 50 µm. (I and J) Cerebral cortical tissues from wild-type mice were lysed with RIPA buffer, and the RIPA lysates were used for immunoprecipitation with each HJ antibody. ApoE proteins left in the supernatant of postimmunoprecipitation (Post-IP) solution (I) and eluted from protein G–Sepharose beads (J) were detected with a polyclonal anti-apoE antibody (EMD Millipore).

    Journal: The Journal of Experimental Medicine

    Article Title: Anti-apoE immunotherapy inhibits amyloid accumulation in a transgenic mouse model of A? amyloidosis

    doi: 10.1084/jem.20121274

    Figure Lengend Snippet: Characterization of anti-apoE antibodies. Four different anti-apoE antibodies were tested for their ability to recognize apoE in Western blotting (A and B), ELISA (C), immunohistochemistry (D–H), and immunoprecipitation (I). (A and B) Cerebral cortical tissues from wild-type and apoe KO mice were lysed with RIPA buffer and equal amounts of total proteins were loaded to each well. In B, the membranes shown in A were overexposed to show a weak apoE band in the membranes probed with HJ6.1, HJ6.2, and HJ6.4 antibody. (C) ELISA plates were coated with anti-apoE antibody (WUE4), and each biotinylated HJ antibody was used as a detection antibody. Optical density at 650 nm was measured with a series of different apoe concentrations. (D–G) Biotinylated HJ antibody was used as a primary antibody in the immunohistochemical staining with APPswe/PS1ΔE9 mice brain tissues. Bar, 50 µm. (H) Biotinylated HJ6.3 antibody was used as a primary antibody in the immunohistochemical staining with brain tissues from apoE-deficient APPswe/PS1ΔE9 mice. Bar, 50 µm. (I and J) Cerebral cortical tissues from wild-type mice were lysed with RIPA buffer, and the RIPA lysates were used for immunoprecipitation with each HJ antibody. ApoE proteins left in the supernatant of postimmunoprecipitation (Post-IP) solution (I) and eluted from protein G–Sepharose beads (J) were detected with a polyclonal anti-apoE antibody (EMD Millipore).

    Article Snippet: Cerebral cortical tissues from wild-type and apoe KO mice were lysed with RIPA lysis buffer (1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 25 mM Tris-HCl, and 150 mM NaCl) in the presence of 1X protease inhibitor mixture (Roche).

    Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Immunoprecipitation, Mouse Assay, Staining

    Nsp12 forms dimers under oxidizing conditions. ( A ) and ( B ) Nsp12 forms dimers, and dimer formation is mediated by covalent disulfide bonds. 2-mercaptoethanol (ß-ME) and dithiothreitol (DTT) block cysteine-mediated dimerization. The N protein was used as a positive control. ( C ) Nsp12 homodimerization due to exposure to the oxidizing extracellular environment after cell lysis. HEK293 T cells were seeded in twelve-well plates and transfected with 2 μg of pCMV-HA-N and ( D ) pCMV-HA-Nsp12 plasmids; at 24 hpt, the cells were treated with RIPA lysis buffer with or without 20 mM N-ethylmaleimide. The lysates were also treated with 4x loading buffer with or without β-ME. ( E ) Nsp12 is not incorporated into virions. MARC-145 cells were infected with PRRSV-HuN4 at an MOI of 0.01; 72 h later, the supernatants were collected and pelleted twice through a 30% sucrose cushion, after which the purified virions were fractionated using 10% to 35% sucrose cushions. Samples were collected from the top to the bottom and analyzed by Western blotting. These experiments were performed three times, and a representative result is shown.

    Journal: Emerging Microbes & Infections

    Article Title: The Nsp12-coding region of type 2 PRRSV is required for viral subgenomic mRNA synthesis

    doi: 10.1080/22221751.2019.1679010

    Figure Lengend Snippet: Nsp12 forms dimers under oxidizing conditions. ( A ) and ( B ) Nsp12 forms dimers, and dimer formation is mediated by covalent disulfide bonds. 2-mercaptoethanol (ß-ME) and dithiothreitol (DTT) block cysteine-mediated dimerization. The N protein was used as a positive control. ( C ) Nsp12 homodimerization due to exposure to the oxidizing extracellular environment after cell lysis. HEK293 T cells were seeded in twelve-well plates and transfected with 2 μg of pCMV-HA-N and ( D ) pCMV-HA-Nsp12 plasmids; at 24 hpt, the cells were treated with RIPA lysis buffer with or without 20 mM N-ethylmaleimide. The lysates were also treated with 4x loading buffer with or without β-ME. ( E ) Nsp12 is not incorporated into virions. MARC-145 cells were infected with PRRSV-HuN4 at an MOI of 0.01; 72 h later, the supernatants were collected and pelleted twice through a 30% sucrose cushion, after which the purified virions were fractionated using 10% to 35% sucrose cushions. Samples were collected from the top to the bottom and analyzed by Western blotting. These experiments were performed three times, and a representative result is shown.

    Article Snippet: At 48 h posttransfection (hpt), the cells were collected, washed once with PBS and lysed in RIPA Lysis Buffer containing a protease inhibitor cocktail (Roche).

    Techniques: Blocking Assay, Positive Control, Lysis, Transfection, Infection, Purification, Western Blot