Structured Review

Roche radioimmunoprecipitation assay buffer
Identification of endogenous ubiquitylation sites in murine tissues by mass spectrometry. A , Frozen tissues were cryogrinded and proteins were solubilized in modified <t>radioimmunoprecipitation</t> assay buffer. Proteins were digested using trypsin, and di-glycine modified peptides were enriched using two different di-glycine-lysine-specific monoclonal antibodies. Enriched peptides were fractionated using strong-cation exchange chromatography and analyzed by mass spectrometry. B , The bar chart illustrates the total number of ubiquitylation sites and the number of tissue-specific ubiquitylation sites identified in each tissue. The number of ubiquitylation sites identified from all tissues (except liver) is combined from two independent replicate experiments. The number of ubiquitylation sites identified from liver tissue is combined from three independent replicate experiments (indicated by an asterisk). C , Number of experiments in which individual sites were found. Over 70% of the reported sites were found in two or more experiments. D , Venn diagram illustrating the experimental overlap between the three replicate experiments performed from liver tissue.
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Images

1) Product Images from "Proteomic Analyses Reveal Divergent Ubiquitylation Site Patterns in Murine Tissues *"

Article Title: Proteomic Analyses Reveal Divergent Ubiquitylation Site Patterns in Murine Tissues *

Journal: Molecular & Cellular Proteomics : MCP

doi: 10.1074/mcp.M112.017905

Identification of endogenous ubiquitylation sites in murine tissues by mass spectrometry. A , Frozen tissues were cryogrinded and proteins were solubilized in modified radioimmunoprecipitation assay buffer. Proteins were digested using trypsin, and di-glycine modified peptides were enriched using two different di-glycine-lysine-specific monoclonal antibodies. Enriched peptides were fractionated using strong-cation exchange chromatography and analyzed by mass spectrometry. B , The bar chart illustrates the total number of ubiquitylation sites and the number of tissue-specific ubiquitylation sites identified in each tissue. The number of ubiquitylation sites identified from all tissues (except liver) is combined from two independent replicate experiments. The number of ubiquitylation sites identified from liver tissue is combined from three independent replicate experiments (indicated by an asterisk). C , Number of experiments in which individual sites were found. Over 70% of the reported sites were found in two or more experiments. D , Venn diagram illustrating the experimental overlap between the three replicate experiments performed from liver tissue.
Figure Legend Snippet: Identification of endogenous ubiquitylation sites in murine tissues by mass spectrometry. A , Frozen tissues were cryogrinded and proteins were solubilized in modified radioimmunoprecipitation assay buffer. Proteins were digested using trypsin, and di-glycine modified peptides were enriched using two different di-glycine-lysine-specific monoclonal antibodies. Enriched peptides were fractionated using strong-cation exchange chromatography and analyzed by mass spectrometry. B , The bar chart illustrates the total number of ubiquitylation sites and the number of tissue-specific ubiquitylation sites identified in each tissue. The number of ubiquitylation sites identified from all tissues (except liver) is combined from two independent replicate experiments. The number of ubiquitylation sites identified from liver tissue is combined from three independent replicate experiments (indicated by an asterisk). C , Number of experiments in which individual sites were found. Over 70% of the reported sites were found in two or more experiments. D , Venn diagram illustrating the experimental overlap between the three replicate experiments performed from liver tissue.

Techniques Used: Mass Spectrometry, Modification, Radio Immunoprecipitation, Chromatography

2) Product Images from "Phosphorylation of VE-cadherin controls endothelial phenotypes via p120-catenin coupling and Rac1 activation"

Article Title: Phosphorylation of VE-cadherin controls endothelial phenotypes via p120-catenin coupling and Rac1 activation

Journal: American Journal of Physiology - Heart and Circulatory Physiology

doi: 10.1152/ajpheart.00650.2010

Knockdown of N-cadherin restores VE-cadherin localization at adherens junctions. A : RFPECs expressing VEC-WT was treated with N-cadherin small interfering RNA (siRNA). Forty eight hours later, total cell lysates were harvested with radioimmunoprecipitation
Figure Legend Snippet: Knockdown of N-cadherin restores VE-cadherin localization at adherens junctions. A : RFPECs expressing VEC-WT was treated with N-cadherin small interfering RNA (siRNA). Forty eight hours later, total cell lysates were harvested with radioimmunoprecipitation

Techniques Used: Expressing, Small Interfering RNA

3) Product Images from "Phosphorylation of the Herpes Simplex Virus Tegument Protein VP22 Has No Effect on Incorporation of VP22 into the Virus but Is Involved in Optimal Expression and Virion Packaging of ICP0"

Article Title: Phosphorylation of the Herpes Simplex Virus Tegument Protein VP22 Has No Effect on Incorporation of VP22 into the Virus but Is Involved in Optimal Expression and Virion Packaging of ICP0

Journal:

doi: 10.1128/JVI.79.22.14057-14068.2005

The phosphorylation variants of VP22 interact with VP16. (A) Monolayers of Vero cells were either mock infected or infected at a multiplicity of infection of 10 with G22v, G22P−v, G22P+v, or the Δ22 virus (169v) and lysed in radioimmunoprecipitation
Figure Legend Snippet: The phosphorylation variants of VP22 interact with VP16. (A) Monolayers of Vero cells were either mock infected or infected at a multiplicity of infection of 10 with G22v, G22P−v, G22P+v, or the Δ22 virus (169v) and lysed in radioimmunoprecipitation

Techniques Used: Infection

4) Product Images from "Mutation of YMYL in the Nipah Virus Matrix Protein Abrogates Budding and Alters Subcellular Localization ▿"

Article Title: Mutation of YMYL in the Nipah Virus Matrix Protein Abrogates Budding and Alters Subcellular Localization ▿

Journal: Journal of Virology

doi: 10.1128/JVI.01743-06

NiV M can bud from transfected cells and form virus-like particles. (a) HEK 293T cells were transfected as indicated with an expression plasmid encoding GFP (2 μg), HA NiV M (2 μg), NiV G (0.5 μg), or NiV F (1 μg). At 48 hours posttransfection, the culture medium was harvested and centrifuged through a 20% sucrose cushion to pellet VLPs, and the transfected cells were lysed in radioimmunoprecipitation assay buffer. Western blotting (IB) of lysates and the purified culture medium was performed with anti-GFP and anti-HA antibodies. (b) Purified HA NiV M VLPs were treated with TPCK-trypsin in the presence or absence of 1% Triton X-100 for 1 h at 37°C. They were then visualized by Western blotting with an anti-HA antibody. (c and d) Representative transmission electron micrographs of NiV VLPs produced by transfection of M alone (c) or M, G, and F (d), stained with 1% phosphotungstic acid, pH 7.0. Arrowheads indicate the studded appearance of NiV glycoproteins, G and/or F, at the VLP surface.
Figure Legend Snippet: NiV M can bud from transfected cells and form virus-like particles. (a) HEK 293T cells were transfected as indicated with an expression plasmid encoding GFP (2 μg), HA NiV M (2 μg), NiV G (0.5 μg), or NiV F (1 μg). At 48 hours posttransfection, the culture medium was harvested and centrifuged through a 20% sucrose cushion to pellet VLPs, and the transfected cells were lysed in radioimmunoprecipitation assay buffer. Western blotting (IB) of lysates and the purified culture medium was performed with anti-GFP and anti-HA antibodies. (b) Purified HA NiV M VLPs were treated with TPCK-trypsin in the presence or absence of 1% Triton X-100 for 1 h at 37°C. They were then visualized by Western blotting with an anti-HA antibody. (c and d) Representative transmission electron micrographs of NiV VLPs produced by transfection of M alone (c) or M, G, and F (d), stained with 1% phosphotungstic acid, pH 7.0. Arrowheads indicate the studded appearance of NiV glycoproteins, G and/or F, at the VLP surface.

Techniques Used: Transfection, Expressing, Plasmid Preparation, Radio Immunoprecipitation, Western Blot, Purification, Transmission Assay, Produced, Staining

5) Product Images from "The Varicella-Zoster Virus Immediate-Early 63 protein affects chromatin controlled gene transcription in a cell-type dependent manner"

Article Title: The Varicella-Zoster Virus Immediate-Early 63 protein affects chromatin controlled gene transcription in a cell-type dependent manner

Journal: BMC Molecular Biology

doi: 10.1186/1471-2199-8-99

Relative expression of selected cellular genes in MeWo-IE63 cells versus control cells . (A) Cells were lysed in radioimmunoprecipitation assay buffer and used for immunoblotting with mouse monoclonal antibody to the ORF63 protein or with rabbit polyclonal antibody to the GFP. (B) mRNA levels (TRAF1, HSPA6, MCP-1, and HLA-DRB1) were determined by quantitative real-time PCR and normalized using the β2-microglobuline transcripts. Experiments were done at least in triplicate. Differences (n-fold) between samples were calculated using the standard-curve method and the 2 -ΔCt method. ρ-values were calculated using the graphpad quickcalcs software [59]: *, significantly different from control (Inv ; p-value
Figure Legend Snippet: Relative expression of selected cellular genes in MeWo-IE63 cells versus control cells . (A) Cells were lysed in radioimmunoprecipitation assay buffer and used for immunoblotting with mouse monoclonal antibody to the ORF63 protein or with rabbit polyclonal antibody to the GFP. (B) mRNA levels (TRAF1, HSPA6, MCP-1, and HLA-DRB1) were determined by quantitative real-time PCR and normalized using the β2-microglobuline transcripts. Experiments were done at least in triplicate. Differences (n-fold) between samples were calculated using the standard-curve method and the 2 -ΔCt method. ρ-values were calculated using the graphpad quickcalcs software [59]: *, significantly different from control (Inv ; p-value

Techniques Used: Expressing, Radio Immunoprecipitation, Real-time Polymerase Chain Reaction, Software

VZV ORF 63 activity and expression by Lentivirus . HeLa cells were infected with the lentivirus (Lenti-IE63, Lenti-Inv, Lenti-IE63-S224/T222A, Lenti-IE63-Full) in order to generate cell lines that stably express the protein IE63 wild-type (HeLa-IE63), in inverted orientation (HeLa-Inv) or mutated (HeLa-S224/T222A, HeLa-Full). One week after infection, cells were harvested. (A) Cells were lysed in radioimmunoprecipitation assay buffer and used for immunoblotting with mouse monoclonal antibody to the ORF63 protein or with rabbit polyclonal antibody to the EGFP. (B) Forty-eight hours post-seeding, immunostaining analysis was carried out using a monoclonal antibody (9A12) directed against IE63. Secondary antibody used is conjugated with Texas Red. (C) HeLa cells (HeLa-IE63, and HeLa-Inv) were transfected with 1 μg of pPol-Luc. 24 hours post-transfection, cells were harvested and the reporter gene activity was measured. Results are presented as a percentage of stimulation with respect to the basal expression of the promoter (= 100%). Data from luciferase assays were collected from six independent transfection experiments. ρ-values were calculated using the graphpad quickcalcs software [59]: *, significantly different from control (p-value
Figure Legend Snippet: VZV ORF 63 activity and expression by Lentivirus . HeLa cells were infected with the lentivirus (Lenti-IE63, Lenti-Inv, Lenti-IE63-S224/T222A, Lenti-IE63-Full) in order to generate cell lines that stably express the protein IE63 wild-type (HeLa-IE63), in inverted orientation (HeLa-Inv) or mutated (HeLa-S224/T222A, HeLa-Full). One week after infection, cells were harvested. (A) Cells were lysed in radioimmunoprecipitation assay buffer and used for immunoblotting with mouse monoclonal antibody to the ORF63 protein or with rabbit polyclonal antibody to the EGFP. (B) Forty-eight hours post-seeding, immunostaining analysis was carried out using a monoclonal antibody (9A12) directed against IE63. Secondary antibody used is conjugated with Texas Red. (C) HeLa cells (HeLa-IE63, and HeLa-Inv) were transfected with 1 μg of pPol-Luc. 24 hours post-transfection, cells were harvested and the reporter gene activity was measured. Results are presented as a percentage of stimulation with respect to the basal expression of the promoter (= 100%). Data from luciferase assays were collected from six independent transfection experiments. ρ-values were calculated using the graphpad quickcalcs software [59]: *, significantly different from control (p-value

Techniques Used: Activity Assay, Expressing, Infection, Stable Transfection, Radio Immunoprecipitation, Immunostaining, Transfection, Luciferase, Software, Significance Assay

Related Articles

Clone Assay:

Article Title: The Zebra fish cassiopeia Mutant Reveals that SIL Is Required for Mitotic Spindle Organization ▿ Mutant Reveals that SIL Is Required for Mitotic Spindle Organization ▿ §
Article Snippet: A sil fragment corresponding to the C-terminal 348 amino acids was generated and cloned into pDONR201 with Gateway BP reaction mix (Invitrogen). .. For immunoblotting, HEK293T cells were lysed with radioimmunoprecipitation assay buffer supplemented with complete protease inhibitors (Roche).

Centrifugation:

Article Title: Mutation of YMYL in the Nipah Virus Matrix Protein Abrogates Budding and Alters Subcellular Localization ▿
Article Snippet: After centrifugation, the supernatant was aspirated and the pelleted VLPs were resuspended in NTE buffer. .. Transfected cells were washed briefly with phosphate-buffered saline and lysed in radioimmunoprecipitation assay buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 0.1% sodium dodecyl sulfate [SDS], 0.5% deoxycholate, 1% NP-40) supplemented with protease inhibitor cocktail (Complete; Roche, Mannheim, Germany).

Article Title: Loss of the Ubiquitin-conjugating Enzyme UBE2W Results in Susceptibility to Early Postnatal Lethality and Defects in Skin, Immune, and Male Reproductive Systems *
Article Snippet: .. Protein lysates from different organs were generated by lysis in radioimmunoprecipitation assay buffer containing protease inhibitors (Complete Mini, Roche Diagnostics) followed by sonication and centrifugation. .. The supernatants were collected, total protein concentration was determined using the BCA method (Pierce), and supernatants were then stored at −80 °C.

Article Title:
Article Snippet: .. After brief centrifugation, cell pellets were lysed in radioimmunoprecipitation assay buffer containing a commercial protease inhibitor mix (Roche Applied Science, Indianapolis, IN) and phosphatase inhibitor (50 mM sodium fluoride and 10 mM sodium orthovanadate). .. After quantification by Bradford protein assay (Bio-Rad Laboratories, Hercules, CA), the proteins were resolved by 10% SDS-PAGE and transferred onto a nitrocellulose membrane (Bio-Rad Laboratories).

Autoradiography:

Article Title: Loss of the Ubiquitin-conjugating Enzyme UBE2W Results in Susceptibility to Early Postnatal Lethality and Defects in Skin, Immune, and Male Reproductive Systems *
Article Snippet: Protein lysates from different organs were generated by lysis in radioimmunoprecipitation assay buffer containing protease inhibitors (Complete Mini, Roche Diagnostics) followed by sonication and centrifugation. .. Bound primary antibodies were visualized by incubation with a peroxidase-conjugated anti-mouse or anti-rabbit secondary antibody (1:10,000; Jackson ImmunoResearch Laboratories, West Grove, PA) followed by treatment with the ECL Plus reagent (Western Lighting, PerkinElmer Life Sciences) and exposure to autoradiography films.

Article Title: Phosphorylation of the Herpes Simplex Virus Tegument Protein VP22 Has No Effect on Incorporation of VP22 into the Virus but Is Involved in Optimal Expression and Virion Packaging of ICP0
Article Snippet: Radioactive labeling was conducted by the addition of 100 μCi of [32 P]orthophosphate (Amersham) per dish, and the cells were incubated at 37°C for a further 13 h. After washing off the unincorporated label with PBS, proteins were extracted with 1 ml of radioimmunoprecipitation assay buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.1% SDS, 1% Na deoxycholate, 0.5% Triton X-100) containing protease inhibitors (Miniprotease inhibitors; Roche Diagnostics Ltd.). .. Proteins from the soluble fraction were immunoprecipitated with the anti-VP22 polyclonal antibody AGV031, and after extensive washing of the protein A Sepharose beads (Amersham), proteins were analyzed by SDS-PAGE and autoradiography.

Blocking Assay:

Article Title:
Article Snippet: After brief centrifugation, cell pellets were lysed in radioimmunoprecipitation assay buffer containing a commercial protease inhibitor mix (Roche Applied Science, Indianapolis, IN) and phosphatase inhibitor (50 mM sodium fluoride and 10 mM sodium orthovanadate). .. After blocking the membrane with 5% nonfat dry milk prepared in Tris-buffered saline + 0.1% Tween-20, the membrane was incubated with the desired primary antibody according to the manufacturer’s directions at 4°C overnight.

Electrophoresis:

Article Title: Autophagy Promotes Oligodendrocyte Survival and Function following Dysmyelination in a Long-Lived Myelin Mutant
Article Snippet: Protein was extracted using radioimmunoprecipitation assay buffer: 20mM Tris, 150mM NaCl, 0.1% SDS, 1% NP40, and protease inhibitor (Complete, Roche) and quantified using the Quant-iT protein detection kit with the Qubit flourometer according to manufacturer’s protocols (Invitrogen). .. A total of 20 µg of each sample was run on a 12% acrylamide gel and blotted onto Immobilon-P membranes (Millipore) using the Mini-PROTEAN electrophoresis system (Bio-Rad).

Acrylamide Gel Assay:

Article Title: Autophagy Promotes Oligodendrocyte Survival and Function following Dysmyelination in a Long-Lived Myelin Mutant
Article Snippet: Protein was extracted using radioimmunoprecipitation assay buffer: 20mM Tris, 150mM NaCl, 0.1% SDS, 1% NP40, and protease inhibitor (Complete, Roche) and quantified using the Quant-iT protein detection kit with the Qubit flourometer according to manufacturer’s protocols (Invitrogen). .. A total of 20 µg of each sample was run on a 12% acrylamide gel and blotted onto Immobilon-P membranes (Millipore) using the Mini-PROTEAN electrophoresis system (Bio-Rad).

Incubation:

Article Title: BMP4 regulates the hematopoietic stem cell niche
Article Snippet: For detection of BMP4 in bone marrow, cell extracts were prepared by sonication in radioimmunoprecipitation assay buffer (50 mM Tris [tris(hydroxymethyl)aminomethane], pH 8; 150 mM NaCl; 0.1% sodium dodecyl sulfate [SDS], 1% nonidet P-40, 0.5% sodium deoxycholate, 1× Roche mini complete protease inhibitor cocktail [Roche]). .. Samples were then incubated on ice for 20 minutes and centrifuged to remove cellular debris.

Article Title: Phosphorylation of VE-cadherin controls endothelial phenotypes via p120-catenin coupling and Rac1 activation
Article Snippet: Cells were washed twice with ice-cold PBS and lysed in radioimmunoprecipitation assay buffer containing 50 mM Tris·HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40 (NP-40), 0.5% sodium deoxycholate, 0.1% SDS, and 1× complete protease inhibitor mixture (Roche). .. For immunoprecipitation assay, total cell lysates were harvested with HEPES lysis buffer with NP-40 containing 50 mM HEPES (pH 7.4), 150 mM NaCl, 1% NP-40, 5 mM EDTA, and 1× complete protease inhibitor mixture (Roche) and incubated with the appropriate antibody and Dynabeads Protein G (Invitrogen).

Article Title: Loss of the Ubiquitin-conjugating Enzyme UBE2W Results in Susceptibility to Early Postnatal Lethality and Defects in Skin, Immune, and Male Reproductive Systems *
Article Snippet: Protein lysates from different organs were generated by lysis in radioimmunoprecipitation assay buffer containing protease inhibitors (Complete Mini, Roche Diagnostics) followed by sonication and centrifugation. .. 40 μg of total proteins per sample were resolved by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and corresponding polyvinylidene difluoride membranes were incubated overnight at 4 °C with the following primary antibodies: rabbit anti-UBE2W (1:1,000; 15920-1-AP, Protein Tech Group), mouse anti-GAPDH (1:10,000; MAB374, Millipore), rabbit anti-Histone H3 (1:10,000; 9715, Cell Signaling Technology), rabbit anti-Gαi-2 (1:1,000; sc-4222, Santa Cruz Biotechnology), mouse anti-Hsp90 (1:1,000; Enzo Life Sciences), and goat anti-protamine-2 (1:250; sc-23104, Santa Cruz Biotechnology).

Article Title:
Article Snippet: After brief centrifugation, cell pellets were lysed in radioimmunoprecipitation assay buffer containing a commercial protease inhibitor mix (Roche Applied Science, Indianapolis, IN) and phosphatase inhibitor (50 mM sodium fluoride and 10 mM sodium orthovanadate). .. After blocking the membrane with 5% nonfat dry milk prepared in Tris-buffered saline + 0.1% Tween-20, the membrane was incubated with the desired primary antibody according to the manufacturer’s directions at 4°C overnight.

Article Title: Autophagy Promotes Oligodendrocyte Survival and Function following Dysmyelination in a Long-Lived Myelin Mutant
Article Snippet: Protein was extracted using radioimmunoprecipitation assay buffer: 20mM Tris, 150mM NaCl, 0.1% SDS, 1% NP40, and protease inhibitor (Complete, Roche) and quantified using the Quant-iT protein detection kit with the Qubit flourometer according to manufacturer’s protocols (Invitrogen). .. Membranes were incubated with the following antibodies overnight: rabbit anti-LC3B (1:1000; Cell Signaling Technology) or rabbit anti-p62 (1:10,000, Enzo Life Sciences).

Article Title: Phosphorylation of the Herpes Simplex Virus Tegument Protein VP22 Has No Effect on Incorporation of VP22 into the Virus but Is Involved in Optimal Expression and Virion Packaging of ICP0
Article Snippet: .. Radioactive labeling was conducted by the addition of 100 μCi of [32 P]orthophosphate (Amersham) per dish, and the cells were incubated at 37°C for a further 13 h. After washing off the unincorporated label with PBS, proteins were extracted with 1 ml of radioimmunoprecipitation assay buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.1% SDS, 1% Na deoxycholate, 0.5% Triton X-100) containing protease inhibitors (Miniprotease inhibitors; Roche Diagnostics Ltd.). .. Proteins from the soluble fraction were immunoprecipitated with the anti-VP22 polyclonal antibody AGV031, and after extensive washing of the protein A Sepharose beads (Amersham), proteins were analyzed by SDS-PAGE and autoradiography.

Article Title: The Zebra fish cassiopeia Mutant Reveals that SIL Is Required for Mitotic Spindle Organization ▿ Mutant Reveals that SIL Is Required for Mitotic Spindle Organization ▿ §
Article Snippet: Purified, glutathione S -transferase-tagged protein was used to immunize rabbits according to standard protocols by Covance, Inc. Nitrocellulose carrying ∼1 mg maltose-binding protein-tagged C-terminal SIL was incubated with antiserum and washed, and SIL-specific antibodies were eluted with 100 mM glycine-HCl (pH 2.5). .. For immunoblotting, HEK293T cells were lysed with radioimmunoprecipitation assay buffer supplemented with complete protease inhibitors (Roche).

Infection:

Article Title: Phosphorylation of the Herpes Simplex Virus Tegument Protein VP22 Has No Effect on Incorporation of VP22 into the Virus but Is Involved in Optimal Expression and Virion Packaging of ICP0
Article Snippet: Confluent monolayers of Vero cells in 6-cm- diameter dishes were infected at a multiplicity of infection of 10 PFU per cell and incubated at 37°C. .. Radioactive labeling was conducted by the addition of 100 μCi of [32 P]orthophosphate (Amersham) per dish, and the cells were incubated at 37°C for a further 13 h. After washing off the unincorporated label with PBS, proteins were extracted with 1 ml of radioimmunoprecipitation assay buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.1% SDS, 1% Na deoxycholate, 0.5% Triton X-100) containing protease inhibitors (Miniprotease inhibitors; Roche Diagnostics Ltd.).

Expressing:

Article Title: Mutation of YMYL in the Nipah Virus Matrix Protein Abrogates Budding and Alters Subcellular Localization ▿
Article Snippet: HEK 293T cells were transfected with 3 μg of expression plasmid by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) as outlined in the manufacturer's protocol. .. Transfected cells were washed briefly with phosphate-buffered saline and lysed in radioimmunoprecipitation assay buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 0.1% sodium dodecyl sulfate [SDS], 0.5% deoxycholate, 1% NP-40) supplemented with protease inhibitor cocktail (Complete; Roche, Mannheim, Germany).

Article Title: The Varicella-Zoster Virus Immediate-Early 63 protein affects chromatin controlled gene transcription in a cell-type dependent manner
Article Snippet: .. Western Blot analysis HeLa or MeWo cells expressing or not wild-type or mutated IE63 were lysed in radioimmunoprecipitation assay buffer (10 mM Tris-HCl pH 8, 100 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% SDS) containing complete protease inhibitor cocktail (Roche), and 5 μg of proteins were separated on a 10% SDS-polyacrylamide gel. ..

BIA-KA:

Article Title: BMP4 regulates the hematopoietic stem cell niche
Article Snippet: For detection of BMP4 in bone marrow, cell extracts were prepared by sonication in radioimmunoprecipitation assay buffer (50 mM Tris [tris(hydroxymethyl)aminomethane], pH 8; 150 mM NaCl; 0.1% sodium dodecyl sulfate [SDS], 1% nonidet P-40, 0.5% sodium deoxycholate, 1× Roche mini complete protease inhibitor cocktail [Roche]). .. Protein concentration was determined by bicinchoninic acid (BCA) analysis (Pierce), and 50 μg total protein for each sample was analyzed.

Article Title: Loss of the Ubiquitin-conjugating Enzyme UBE2W Results in Susceptibility to Early Postnatal Lethality and Defects in Skin, Immune, and Male Reproductive Systems *
Article Snippet: Protein lysates from different organs were generated by lysis in radioimmunoprecipitation assay buffer containing protease inhibitors (Complete Mini, Roche Diagnostics) followed by sonication and centrifugation. .. The supernatants were collected, total protein concentration was determined using the BCA method (Pierce), and supernatants were then stored at −80 °C.

Modification:

Article Title: Proteomic Analyses Reveal Divergent Ubiquitylation Site Patterns in Murine Tissues *
Article Snippet: .. The frozen tissue samples were cryogrinded using a Retsch MM 400 ball mill and lysed in modified radioimmunoprecipitation assay buffer (50 mm Tris pH 7.5, 150 mm NaCl, 1 mm EDTA, 1% Nonidet P-40, 0.1% sodium deoxycholate) supplemented with protease inhibitors (Complete protease inhibitor mixture tablets, Roche Diagnostics) and 5 mm N-ethylmaleimide (Sigma). .. MS Sample Preparation Twenty milligrams of protein from tissue lysates were precipitated in acetone and subsequently re-dissolved in denaturation buffer (6 m urea, 2 m thiourea in 10 mm HEPES pH 8.0).

Western Blot:

Article Title: Mutation of YMYL in the Nipah Virus Matrix Protein Abrogates Budding and Alters Subcellular Localization ▿
Article Snippet: Transfected cells were washed briefly with phosphate-buffered saline and lysed in radioimmunoprecipitation assay buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 0.1% sodium dodecyl sulfate [SDS], 0.5% deoxycholate, 1% NP-40) supplemented with protease inhibitor cocktail (Complete; Roche, Mannheim, Germany). .. VLPs, and lysates were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by Western blotting with anti-green fluorescent protein (anti-GFP) (Clontech, Mountain View, CA), anti-HA (Sigma-Aldrich, St. Louis, MO), or anti-VP40 antibodies. (Anti-VP40 monoclonal antibody was kindly provided by Ronald Harty, University of Pennsylvania.)

Article Title: BMP4 regulates the hematopoietic stem cell niche
Article Snippet: Paragraph title: Western analysis ... For detection of BMP4 in bone marrow, cell extracts were prepared by sonication in radioimmunoprecipitation assay buffer (50 mM Tris [tris(hydroxymethyl)aminomethane], pH 8; 150 mM NaCl; 0.1% sodium dodecyl sulfate [SDS], 1% nonidet P-40, 0.5% sodium deoxycholate, 1× Roche mini complete protease inhibitor cocktail [Roche]).

Article Title: Phosphorylation of VE-cadherin controls endothelial phenotypes via p120-catenin coupling and Rac1 activation
Article Snippet: Paragraph title: Western blot analysis and immunoprecipitation. ... Cells were washed twice with ice-cold PBS and lysed in radioimmunoprecipitation assay buffer containing 50 mM Tris·HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40 (NP-40), 0.5% sodium deoxycholate, 0.1% SDS, and 1× complete protease inhibitor mixture (Roche).

Article Title: A functional genetic screen identifies retinoic acid signaling as a target of histone deacetylase inhibitors
Article Snippet: Paragraph title: Western Blotting and Apoptosis Assays. ... Cells were lysed in radioimmunoprecipitation assay buffer (50 mM Tris, pH 8.0/150 mM NaCl/1% Nonidet P-40/0.5% deoxycholic acid/0.1% SDS) supplemented with protease inhibitors (Complete; Roche, Indianapolis, IN) and 0.2 nM PMSF, and proteins were separated on SDS/10–14% polyacrylamide gels.

Article Title: DAPK2 is a novel modulator of TRAIL-induced apoptosis
Article Snippet: Paragraph title: Western blotting ... Proteins were extracted using radioimmunoprecipitation assay buffer (RIPA; 50 mM Tris-HCl (pH 7.4), 0.5% (v/v) NP-40, 150 mM NaCl, 1 mM EDTA, 1 mM Na3 VO4 and ‘cOmplete and Mini, EDTA-free protease inhibitor cocktail', the latter used as instructed by the manufacturer; Roche, Mannheim, Germany).

Article Title: Loss of the Ubiquitin-conjugating Enzyme UBE2W Results in Susceptibility to Early Postnatal Lethality and Defects in Skin, Immune, and Male Reproductive Systems *
Article Snippet: Paragraph title: Western Blotting ... Protein lysates from different organs were generated by lysis in radioimmunoprecipitation assay buffer containing protease inhibitors (Complete Mini, Roche Diagnostics) followed by sonication and centrifugation.

Article Title:
Article Snippet: Paragraph title: Western Blotting. ... After brief centrifugation, cell pellets were lysed in radioimmunoprecipitation assay buffer containing a commercial protease inhibitor mix (Roche Applied Science, Indianapolis, IN) and phosphatase inhibitor (50 mM sodium fluoride and 10 mM sodium orthovanadate).

Article Title: The Varicella-Zoster Virus Immediate-Early 63 protein affects chromatin controlled gene transcription in a cell-type dependent manner
Article Snippet: .. Western Blot analysis HeLa or MeWo cells expressing or not wild-type or mutated IE63 were lysed in radioimmunoprecipitation assay buffer (10 mM Tris-HCl pH 8, 100 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% SDS) containing complete protease inhibitor cocktail (Roche), and 5 μg of proteins were separated on a 10% SDS-polyacrylamide gel. ..

Article Title: Autophagy Promotes Oligodendrocyte Survival and Function following Dysmyelination in a Long-Lived Myelin Mutant
Article Snippet: Paragraph title: Western blot ... Protein was extracted using radioimmunoprecipitation assay buffer: 20mM Tris, 150mM NaCl, 0.1% SDS, 1% NP40, and protease inhibitor (Complete, Roche) and quantified using the Quant-iT protein detection kit with the Qubit flourometer according to manufacturer’s protocols (Invitrogen).

Article Title: The Zebra fish cassiopeia Mutant Reveals that SIL Is Required for Mitotic Spindle Organization ▿ Mutant Reveals that SIL Is Required for Mitotic Spindle Organization ▿ §
Article Snippet: Paragraph title: Western blotting. ... Protein lysates were extracted from 24- to 30-hpf zebrafish embryos with radioimmunoprecipitation assay buffer supplemented with complete protease inhibitor tablets (Roche).

Article Title: Hypoxia and Glucocorticoid Signaling Converge to Regulate Macrophage Migration Inhibitory Factor Gene Expression
Article Snippet: Paragraph title: Western blotting ... For whole cell lysates, cells were harvested and lysed in radioimmunoprecipitation assay buffer (100 m M Tris [pH 7.4], 150 m M NaCl, 1% Nonidet P40, 2.5% sodium deoxycholate, and 1 m M EDTA) with Complete protease inhibitor (Roche, Indianapolis, IN).

Article Title: HIV Vpu Interferes with NF-κB Activity but Not with Interferon Regulatory Factor 3
Article Snippet: Cells were lysed in radioimmunoprecipitation assay buffer supplemented with complete protease inhibitor (Roche). .. Western blot signals were visualized using SuperSignal West Pico or Femto (Pierce) on a Fluorochem E System protein simple machine.

Article Title: The Zebra fish cassiopeia Mutant Reveals that SIL Is Required for Mitotic Spindle Organization ▿ Mutant Reveals that SIL Is Required for Mitotic Spindle Organization ▿ §
Article Snippet: For immunoblotting, HEK293T cells were lysed with radioimmunoprecipitation assay buffer supplemented with complete protease inhibitors (Roche). .. Standard techniques were used for Western blotting with 1:2,000 antihemagglutinin (anti-HA) (12CA5; Roche) or 1:1,000 anti-SIL.

Transfection:

Article Title: Mutation of YMYL in the Nipah Virus Matrix Protein Abrogates Budding and Alters Subcellular Localization ▿
Article Snippet: .. Transfected cells were washed briefly with phosphate-buffered saline and lysed in radioimmunoprecipitation assay buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 0.1% sodium dodecyl sulfate [SDS], 0.5% deoxycholate, 1% NP-40) supplemented with protease inhibitor cocktail (Complete; Roche, Mannheim, Germany). .. VLPs, and lysates were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by Western blotting with anti-green fluorescent protein (anti-GFP) (Clontech, Mountain View, CA), anti-HA (Sigma-Aldrich, St. Louis, MO), or anti-VP40 antibodies. (Anti-VP40 monoclonal antibody was kindly provided by Ronald Harty, University of Pennsylvania.)

Protease Inhibitor:

Article Title: Mutation of YMYL in the Nipah Virus Matrix Protein Abrogates Budding and Alters Subcellular Localization ▿
Article Snippet: .. Transfected cells were washed briefly with phosphate-buffered saline and lysed in radioimmunoprecipitation assay buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 0.1% sodium dodecyl sulfate [SDS], 0.5% deoxycholate, 1% NP-40) supplemented with protease inhibitor cocktail (Complete; Roche, Mannheim, Germany). .. VLPs, and lysates were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by Western blotting with anti-green fluorescent protein (anti-GFP) (Clontech, Mountain View, CA), anti-HA (Sigma-Aldrich, St. Louis, MO), or anti-VP40 antibodies. (Anti-VP40 monoclonal antibody was kindly provided by Ronald Harty, University of Pennsylvania.)

Article Title: BMP4 regulates the hematopoietic stem cell niche
Article Snippet: .. For detection of BMP4 in bone marrow, cell extracts were prepared by sonication in radioimmunoprecipitation assay buffer (50 mM Tris [tris(hydroxymethyl)aminomethane], pH 8; 150 mM NaCl; 0.1% sodium dodecyl sulfate [SDS], 1% nonidet P-40, 0.5% sodium deoxycholate, 1× Roche mini complete protease inhibitor cocktail [Roche]). .. Samples were then incubated on ice for 20 minutes and centrifuged to remove cellular debris.

Article Title: Phosphorylation of VE-cadherin controls endothelial phenotypes via p120-catenin coupling and Rac1 activation
Article Snippet: .. Cells were washed twice with ice-cold PBS and lysed in radioimmunoprecipitation assay buffer containing 50 mM Tris·HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40 (NP-40), 0.5% sodium deoxycholate, 0.1% SDS, and 1× complete protease inhibitor mixture (Roche). .. Total cell lysates were subjected to SDS-PAGE, followed by immunoblotting.

Article Title: DAPK2 is a novel modulator of TRAIL-induced apoptosis
Article Snippet: .. Proteins were extracted using radioimmunoprecipitation assay buffer (RIPA; 50 mM Tris-HCl (pH 7.4), 0.5% (v/v) NP-40, 150 mM NaCl, 1 mM EDTA, 1 mM Na3 VO4 and ‘cOmplete and Mini, EDTA-free protease inhibitor cocktail', the latter used as instructed by the manufacturer; Roche, Mannheim, Germany). ..

Article Title:
Article Snippet: .. After brief centrifugation, cell pellets were lysed in radioimmunoprecipitation assay buffer containing a commercial protease inhibitor mix (Roche Applied Science, Indianapolis, IN) and phosphatase inhibitor (50 mM sodium fluoride and 10 mM sodium orthovanadate). .. After quantification by Bradford protein assay (Bio-Rad Laboratories, Hercules, CA), the proteins were resolved by 10% SDS-PAGE and transferred onto a nitrocellulose membrane (Bio-Rad Laboratories).

Article Title: The Varicella-Zoster Virus Immediate-Early 63 protein affects chromatin controlled gene transcription in a cell-type dependent manner
Article Snippet: .. Western Blot analysis HeLa or MeWo cells expressing or not wild-type or mutated IE63 were lysed in radioimmunoprecipitation assay buffer (10 mM Tris-HCl pH 8, 100 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% SDS) containing complete protease inhibitor cocktail (Roche), and 5 μg of proteins were separated on a 10% SDS-polyacrylamide gel. ..

Article Title: Proteomic Analyses Reveal Divergent Ubiquitylation Site Patterns in Murine Tissues *
Article Snippet: .. The frozen tissue samples were cryogrinded using a Retsch MM 400 ball mill and lysed in modified radioimmunoprecipitation assay buffer (50 mm Tris pH 7.5, 150 mm NaCl, 1 mm EDTA, 1% Nonidet P-40, 0.1% sodium deoxycholate) supplemented with protease inhibitors (Complete protease inhibitor mixture tablets, Roche Diagnostics) and 5 mm N-ethylmaleimide (Sigma). .. MS Sample Preparation Twenty milligrams of protein from tissue lysates were precipitated in acetone and subsequently re-dissolved in denaturation buffer (6 m urea, 2 m thiourea in 10 mm HEPES pH 8.0).

Article Title: Autophagy Promotes Oligodendrocyte Survival and Function following Dysmyelination in a Long-Lived Myelin Mutant
Article Snippet: .. Protein was extracted using radioimmunoprecipitation assay buffer: 20mM Tris, 150mM NaCl, 0.1% SDS, 1% NP40, and protease inhibitor (Complete, Roche) and quantified using the Quant-iT protein detection kit with the Qubit flourometer according to manufacturer’s protocols (Invitrogen). .. A total of 20 µg of each sample was run on a 12% acrylamide gel and blotted onto Immobilon-P membranes (Millipore) using the Mini-PROTEAN electrophoresis system (Bio-Rad).

Article Title: The Zebra fish cassiopeia Mutant Reveals that SIL Is Required for Mitotic Spindle Organization ▿ Mutant Reveals that SIL Is Required for Mitotic Spindle Organization ▿ §
Article Snippet: .. Protein lysates were extracted from 24- to 30-hpf zebrafish embryos with radioimmunoprecipitation assay buffer supplemented with complete protease inhibitor tablets (Roche). .. Forty micrograms of total protein was subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose for immunoblotting with polyclonal γ-tubulin (Sigma).

Article Title: Hypoxia and Glucocorticoid Signaling Converge to Regulate Macrophage Migration Inhibitory Factor Gene Expression
Article Snippet: .. For whole cell lysates, cells were harvested and lysed in radioimmunoprecipitation assay buffer (100 m M Tris [pH 7.4], 150 m M NaCl, 1% Nonidet P40, 2.5% sodium deoxycholate, and 1 m M EDTA) with Complete protease inhibitor (Roche, Indianapolis, IN). .. Cell lysates and nuclear extracts were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed with one of the following antibodies: anti-GR α (1:1,000 dilution) (GR α -clone 41; BD Biosciences, San Jose, CA) anti-phosphoGR (1:2,000; New England Biolabs, Ipswich, MA), anti–ATF-1 (1:2,000; Santa Cruz Biotechnology, Santa Cruz, CA), anti-CREB (1:2,000; Santa Cruz Biotechnology), anti HIF-1 α (1:2,000; Santa Cruz Biotechnology), and anti-tubulin (1:2,000; Sigma, St. Louis, MO).

Article Title: HIV Vpu Interferes with NF-κB Activity but Not with Interferon Regulatory Factor 3
Article Snippet: .. Cells were lysed in radioimmunoprecipitation assay buffer supplemented with complete protease inhibitor (Roche). .. Proteins were separated by SDS-PAGE (Invitrogen) and transferred to polyvinylidene difluoride membranes (Pierce).

Cell Culture:

Article Title: Mutation of YMYL in the Nipah Virus Matrix Protein Abrogates Budding and Alters Subcellular Localization ▿
Article Snippet: At 48 hours posttransfection, the transfected cell culture medium was harvested and clarified by centrifugation at 200 × g for 5 min. .. Transfected cells were washed briefly with phosphate-buffered saline and lysed in radioimmunoprecipitation assay buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 0.1% sodium dodecyl sulfate [SDS], 0.5% deoxycholate, 1% NP-40) supplemented with protease inhibitor cocktail (Complete; Roche, Mannheim, Germany).

Article Title: A functional genetic screen identifies retinoic acid signaling as a target of histone deacetylase inhibitors
Article Snippet: Cells were lysed in radioimmunoprecipitation assay buffer (50 mM Tris, pH 8.0/150 mM NaCl/1% Nonidet P-40/0.5% deoxycholic acid/0.1% SDS) supplemented with protease inhibitors (Complete; Roche, Indianapolis, IN) and 0.2 nM PMSF, and proteins were separated on SDS/10–14% polyacrylamide gels. .. To measure apoptosis, cells were plated at a density of 10,000 cells per well in 96-well plates and cultured for 24 h; HDACI were added, and the cells were cultured for another 24 h. The cells were lysed, and apoptosis was detected by using the Apo-ONE assay (Promega), which quantifies caspase 3/7-specific cleavage of a peptide-based substrate into a fluorescent product.

Generated:

Article Title: Loss of the Ubiquitin-conjugating Enzyme UBE2W Results in Susceptibility to Early Postnatal Lethality and Defects in Skin, Immune, and Male Reproductive Systems *
Article Snippet: .. Protein lysates from different organs were generated by lysis in radioimmunoprecipitation assay buffer containing protease inhibitors (Complete Mini, Roche Diagnostics) followed by sonication and centrifugation. .. The supernatants were collected, total protein concentration was determined using the BCA method (Pierce), and supernatants were then stored at −80 °C.

Article Title: The Zebra fish cassiopeia Mutant Reveals that SIL Is Required for Mitotic Spindle Organization ▿ Mutant Reveals that SIL Is Required for Mitotic Spindle Organization ▿ §
Article Snippet: A sil fragment corresponding to the C-terminal 348 amino acids was generated and cloned into pDONR201 with Gateway BP reaction mix (Invitrogen). .. For immunoblotting, HEK293T cells were lysed with radioimmunoprecipitation assay buffer supplemented with complete protease inhibitors (Roche).

Protein Concentration:

Article Title: BMP4 regulates the hematopoietic stem cell niche
Article Snippet: For detection of BMP4 in bone marrow, cell extracts were prepared by sonication in radioimmunoprecipitation assay buffer (50 mM Tris [tris(hydroxymethyl)aminomethane], pH 8; 150 mM NaCl; 0.1% sodium dodecyl sulfate [SDS], 1% nonidet P-40, 0.5% sodium deoxycholate, 1× Roche mini complete protease inhibitor cocktail [Roche]). .. Protein concentration was determined by bicinchoninic acid (BCA) analysis (Pierce), and 50 μg total protein for each sample was analyzed.

Article Title: Loss of the Ubiquitin-conjugating Enzyme UBE2W Results in Susceptibility to Early Postnatal Lethality and Defects in Skin, Immune, and Male Reproductive Systems *
Article Snippet: Protein lysates from different organs were generated by lysis in radioimmunoprecipitation assay buffer containing protease inhibitors (Complete Mini, Roche Diagnostics) followed by sonication and centrifugation. .. The supernatants were collected, total protein concentration was determined using the BCA method (Pierce), and supernatants were then stored at −80 °C.

Sonication:

Article Title: BMP4 regulates the hematopoietic stem cell niche
Article Snippet: .. For detection of BMP4 in bone marrow, cell extracts were prepared by sonication in radioimmunoprecipitation assay buffer (50 mM Tris [tris(hydroxymethyl)aminomethane], pH 8; 150 mM NaCl; 0.1% sodium dodecyl sulfate [SDS], 1% nonidet P-40, 0.5% sodium deoxycholate, 1× Roche mini complete protease inhibitor cocktail [Roche]). .. Samples were then incubated on ice for 20 minutes and centrifuged to remove cellular debris.

Article Title: Loss of the Ubiquitin-conjugating Enzyme UBE2W Results in Susceptibility to Early Postnatal Lethality and Defects in Skin, Immune, and Male Reproductive Systems *
Article Snippet: .. Protein lysates from different organs were generated by lysis in radioimmunoprecipitation assay buffer containing protease inhibitors (Complete Mini, Roche Diagnostics) followed by sonication and centrifugation. .. The supernatants were collected, total protein concentration was determined using the BCA method (Pierce), and supernatants were then stored at −80 °C.

Nucleic Acid Electrophoresis:

Article Title: Mutation of YMYL in the Nipah Virus Matrix Protein Abrogates Budding and Alters Subcellular Localization ▿
Article Snippet: Transfected cells were washed briefly with phosphate-buffered saline and lysed in radioimmunoprecipitation assay buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 0.1% sodium dodecyl sulfate [SDS], 0.5% deoxycholate, 1% NP-40) supplemented with protease inhibitor cocktail (Complete; Roche, Mannheim, Germany). .. VLPs, and lysates were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by Western blotting with anti-green fluorescent protein (anti-GFP) (Clontech, Mountain View, CA), anti-HA (Sigma-Aldrich, St. Louis, MO), or anti-VP40 antibodies. (Anti-VP40 monoclonal antibody was kindly provided by Ronald Harty, University of Pennsylvania.)

Article Title: Loss of the Ubiquitin-conjugating Enzyme UBE2W Results in Susceptibility to Early Postnatal Lethality and Defects in Skin, Immune, and Male Reproductive Systems *
Article Snippet: Protein lysates from different organs were generated by lysis in radioimmunoprecipitation assay buffer containing protease inhibitors (Complete Mini, Roche Diagnostics) followed by sonication and centrifugation. .. 40 μg of total proteins per sample were resolved by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and corresponding polyvinylidene difluoride membranes were incubated overnight at 4 °C with the following primary antibodies: rabbit anti-UBE2W (1:1,000; 15920-1-AP, Protein Tech Group), mouse anti-GAPDH (1:10,000; MAB374, Millipore), rabbit anti-Histone H3 (1:10,000; 9715, Cell Signaling Technology), rabbit anti-Gαi-2 (1:1,000; sc-4222, Santa Cruz Biotechnology), mouse anti-Hsp90 (1:1,000; Enzo Life Sciences), and goat anti-protamine-2 (1:250; sc-23104, Santa Cruz Biotechnology).

Article Title: The Zebra fish cassiopeia Mutant Reveals that SIL Is Required for Mitotic Spindle Organization ▿ Mutant Reveals that SIL Is Required for Mitotic Spindle Organization ▿ §
Article Snippet: Protein lysates were extracted from 24- to 30-hpf zebrafish embryos with radioimmunoprecipitation assay buffer supplemented with complete protease inhibitor tablets (Roche). .. Forty micrograms of total protein was subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose for immunoblotting with polyclonal γ-tubulin (Sigma).

Article Title: Hypoxia and Glucocorticoid Signaling Converge to Regulate Macrophage Migration Inhibitory Factor Gene Expression
Article Snippet: For whole cell lysates, cells were harvested and lysed in radioimmunoprecipitation assay buffer (100 m M Tris [pH 7.4], 150 m M NaCl, 1% Nonidet P40, 2.5% sodium deoxycholate, and 1 m M EDTA) with Complete protease inhibitor (Roche, Indianapolis, IN). .. Cell lysates and nuclear extracts were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed with one of the following antibodies: anti-GR α (1:1,000 dilution) (GR α -clone 41; BD Biosciences, San Jose, CA) anti-phosphoGR (1:2,000; New England Biolabs, Ipswich, MA), anti–ATF-1 (1:2,000; Santa Cruz Biotechnology, Santa Cruz, CA), anti-CREB (1:2,000; Santa Cruz Biotechnology), anti HIF-1 α (1:2,000; Santa Cruz Biotechnology), and anti-tubulin (1:2,000; Sigma, St. Louis, MO).

Article Title: The Zebra fish cassiopeia Mutant Reveals that SIL Is Required for Mitotic Spindle Organization ▿ Mutant Reveals that SIL Is Required for Mitotic Spindle Organization ▿ §
Article Snippet: For immunoblotting, HEK293T cells were lysed with radioimmunoprecipitation assay buffer supplemented with complete protease inhibitors (Roche). .. Thirty micrograms of total protein was subjected to 6% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose.

In Vivo:

Article Title: Phosphorylation of the Herpes Simplex Virus Tegument Protein VP22 Has No Effect on Incorporation of VP22 into the Virus but Is Involved in Optimal Expression and Virion Packaging of ICP0
Article Snippet: Paragraph title: In vivo radiolabeling assay. ... Radioactive labeling was conducted by the addition of 100 μCi of [32 P]orthophosphate (Amersham) per dish, and the cells were incubated at 37°C for a further 13 h. After washing off the unincorporated label with PBS, proteins were extracted with 1 ml of radioimmunoprecipitation assay buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.1% SDS, 1% Na deoxycholate, 0.5% Triton X-100) containing protease inhibitors (Miniprotease inhibitors; Roche Diagnostics Ltd.).

Radioactivity:

Article Title: Phosphorylation of the Herpes Simplex Virus Tegument Protein VP22 Has No Effect on Incorporation of VP22 into the Virus but Is Involved in Optimal Expression and Virion Packaging of ICP0
Article Snippet: Paragraph title: In vivo radiolabeling assay. ... Radioactive labeling was conducted by the addition of 100 μCi of [32 P]orthophosphate (Amersham) per dish, and the cells were incubated at 37°C for a further 13 h. After washing off the unincorporated label with PBS, proteins were extracted with 1 ml of radioimmunoprecipitation assay buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.1% SDS, 1% Na deoxycholate, 0.5% Triton X-100) containing protease inhibitors (Miniprotease inhibitors; Roche Diagnostics Ltd.).

Mutagenesis:

Article Title: BMP4 regulates the hematopoietic stem cell niche
Article Snippet: For detection of BMP4 in bone marrow, cell extracts were prepared by sonication in radioimmunoprecipitation assay buffer (50 mM Tris [tris(hydroxymethyl)aminomethane], pH 8; 150 mM NaCl; 0.1% sodium dodecyl sulfate [SDS], 1% nonidet P-40, 0.5% sodium deoxycholate, 1× Roche mini complete protease inhibitor cocktail [Roche]). .. For assessment of phosphorylated receptor Smads (Smad1, Smad5, and Smad8) in c-kit+ bone marrow cells, equal numbers of cells were sorted from pairs of WT and mutant mice (2.4 × 105 cells per mouse and 3.2 × 105 cells per mouse in 2 independent experiments).

Labeling:

Article Title: Phosphorylation of the Herpes Simplex Virus Tegument Protein VP22 Has No Effect on Incorporation of VP22 into the Virus but Is Involved in Optimal Expression and Virion Packaging of ICP0
Article Snippet: .. Radioactive labeling was conducted by the addition of 100 μCi of [32 P]orthophosphate (Amersham) per dish, and the cells were incubated at 37°C for a further 13 h. After washing off the unincorporated label with PBS, proteins were extracted with 1 ml of radioimmunoprecipitation assay buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.1% SDS, 1% Na deoxycholate, 0.5% Triton X-100) containing protease inhibitors (Miniprotease inhibitors; Roche Diagnostics Ltd.). .. Proteins from the soluble fraction were immunoprecipitated with the anti-VP22 polyclonal antibody AGV031, and after extensive washing of the protein A Sepharose beads (Amersham), proteins were analyzed by SDS-PAGE and autoradiography.

Mouse Assay:

Article Title: BMP4 regulates the hematopoietic stem cell niche
Article Snippet: For detection of BMP4 in bone marrow, cell extracts were prepared by sonication in radioimmunoprecipitation assay buffer (50 mM Tris [tris(hydroxymethyl)aminomethane], pH 8; 150 mM NaCl; 0.1% sodium dodecyl sulfate [SDS], 1% nonidet P-40, 0.5% sodium deoxycholate, 1× Roche mini complete protease inhibitor cocktail [Roche]). .. For assessment of phosphorylated receptor Smads (Smad1, Smad5, and Smad8) in c-kit+ bone marrow cells, equal numbers of cells were sorted from pairs of WT and mutant mice (2.4 × 105 cells per mouse and 3.2 × 105 cells per mouse in 2 independent experiments).

Article Title: Proteomic Analyses Reveal Divergent Ubiquitylation Site Patterns in Murine Tissues *
Article Snippet: Tissues Mouse tissues were dissected from C57BL/6 mice, rinsed with PBS and frozen immediately in liquid nitrogen. .. The frozen tissue samples were cryogrinded using a Retsch MM 400 ball mill and lysed in modified radioimmunoprecipitation assay buffer (50 mm Tris pH 7.5, 150 mm NaCl, 1 mm EDTA, 1% Nonidet P-40, 0.1% sodium deoxycholate) supplemented with protease inhibitors (Complete protease inhibitor mixture tablets, Roche Diagnostics) and 5 mm N-ethylmaleimide (Sigma).

Bradford Protein Assay:

Article Title:
Article Snippet: After brief centrifugation, cell pellets were lysed in radioimmunoprecipitation assay buffer containing a commercial protease inhibitor mix (Roche Applied Science, Indianapolis, IN) and phosphatase inhibitor (50 mM sodium fluoride and 10 mM sodium orthovanadate). .. After quantification by Bradford protein assay (Bio-Rad Laboratories, Hercules, CA), the proteins were resolved by 10% SDS-PAGE and transferred onto a nitrocellulose membrane (Bio-Rad Laboratories).

Purification:

Article Title: The Zebra fish cassiopeia Mutant Reveals that SIL Is Required for Mitotic Spindle Organization ▿ Mutant Reveals that SIL Is Required for Mitotic Spindle Organization ▿ §
Article Snippet: Purified, glutathione S -transferase-tagged protein was used to immunize rabbits according to standard protocols by Covance, Inc. Nitrocellulose carrying ∼1 mg maltose-binding protein-tagged C-terminal SIL was incubated with antiserum and washed, and SIL-specific antibodies were eluted with 100 mM glycine-HCl (pH 2.5). .. For immunoblotting, HEK293T cells were lysed with radioimmunoprecipitation assay buffer supplemented with complete protease inhibitors (Roche).

SDS Page:

Article Title: Mutation of YMYL in the Nipah Virus Matrix Protein Abrogates Budding and Alters Subcellular Localization ▿
Article Snippet: Transfected cells were washed briefly with phosphate-buffered saline and lysed in radioimmunoprecipitation assay buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 0.1% sodium dodecyl sulfate [SDS], 0.5% deoxycholate, 1% NP-40) supplemented with protease inhibitor cocktail (Complete; Roche, Mannheim, Germany). .. VLPs, and lysates were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by Western blotting with anti-green fluorescent protein (anti-GFP) (Clontech, Mountain View, CA), anti-HA (Sigma-Aldrich, St. Louis, MO), or anti-VP40 antibodies. (Anti-VP40 monoclonal antibody was kindly provided by Ronald Harty, University of Pennsylvania.)

Article Title: Phosphorylation of VE-cadherin controls endothelial phenotypes via p120-catenin coupling and Rac1 activation
Article Snippet: Cells were washed twice with ice-cold PBS and lysed in radioimmunoprecipitation assay buffer containing 50 mM Tris·HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40 (NP-40), 0.5% sodium deoxycholate, 0.1% SDS, and 1× complete protease inhibitor mixture (Roche). .. Total cell lysates were subjected to SDS-PAGE, followed by immunoblotting.

Article Title:
Article Snippet: After brief centrifugation, cell pellets were lysed in radioimmunoprecipitation assay buffer containing a commercial protease inhibitor mix (Roche Applied Science, Indianapolis, IN) and phosphatase inhibitor (50 mM sodium fluoride and 10 mM sodium orthovanadate). .. After quantification by Bradford protein assay (Bio-Rad Laboratories, Hercules, CA), the proteins were resolved by 10% SDS-PAGE and transferred onto a nitrocellulose membrane (Bio-Rad Laboratories).

Article Title: Hypoxia and Glucocorticoid Signaling Converge to Regulate Macrophage Migration Inhibitory Factor Gene Expression
Article Snippet: For whole cell lysates, cells were harvested and lysed in radioimmunoprecipitation assay buffer (100 m M Tris [pH 7.4], 150 m M NaCl, 1% Nonidet P40, 2.5% sodium deoxycholate, and 1 m M EDTA) with Complete protease inhibitor (Roche, Indianapolis, IN). .. Cell lysates and nuclear extracts were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed with one of the following antibodies: anti-GR α (1:1,000 dilution) (GR α -clone 41; BD Biosciences, San Jose, CA) anti-phosphoGR (1:2,000; New England Biolabs, Ipswich, MA), anti–ATF-1 (1:2,000; Santa Cruz Biotechnology, Santa Cruz, CA), anti-CREB (1:2,000; Santa Cruz Biotechnology), anti HIF-1 α (1:2,000; Santa Cruz Biotechnology), and anti-tubulin (1:2,000; Sigma, St. Louis, MO).

Article Title: Phosphorylation of the Herpes Simplex Virus Tegument Protein VP22 Has No Effect on Incorporation of VP22 into the Virus but Is Involved in Optimal Expression and Virion Packaging of ICP0
Article Snippet: Radioactive labeling was conducted by the addition of 100 μCi of [32 P]orthophosphate (Amersham) per dish, and the cells were incubated at 37°C for a further 13 h. After washing off the unincorporated label with PBS, proteins were extracted with 1 ml of radioimmunoprecipitation assay buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.1% SDS, 1% Na deoxycholate, 0.5% Triton X-100) containing protease inhibitors (Miniprotease inhibitors; Roche Diagnostics Ltd.). .. Proteins from the soluble fraction were immunoprecipitated with the anti-VP22 polyclonal antibody AGV031, and after extensive washing of the protein A Sepharose beads (Amersham), proteins were analyzed by SDS-PAGE and autoradiography.

Article Title: HIV Vpu Interferes with NF-κB Activity but Not with Interferon Regulatory Factor 3
Article Snippet: Cells were lysed in radioimmunoprecipitation assay buffer supplemented with complete protease inhibitor (Roche). .. Proteins were separated by SDS-PAGE (Invitrogen) and transferred to polyvinylidene difluoride membranes (Pierce).

Plasmid Preparation:

Article Title: Mutation of YMYL in the Nipah Virus Matrix Protein Abrogates Budding and Alters Subcellular Localization ▿
Article Snippet: HEK 293T cells were transfected with 3 μg of expression plasmid by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) as outlined in the manufacturer's protocol. .. Transfected cells were washed briefly with phosphate-buffered saline and lysed in radioimmunoprecipitation assay buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 0.1% sodium dodecyl sulfate [SDS], 0.5% deoxycholate, 1% NP-40) supplemented with protease inhibitor cocktail (Complete; Roche, Mannheim, Germany).

Radio Immunoprecipitation:

Article Title: Mutation of YMYL in the Nipah Virus Matrix Protein Abrogates Budding and Alters Subcellular Localization ▿
Article Snippet: .. Transfected cells were washed briefly with phosphate-buffered saline and lysed in radioimmunoprecipitation assay buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 0.1% sodium dodecyl sulfate [SDS], 0.5% deoxycholate, 1% NP-40) supplemented with protease inhibitor cocktail (Complete; Roche, Mannheim, Germany). .. VLPs, and lysates were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by Western blotting with anti-green fluorescent protein (anti-GFP) (Clontech, Mountain View, CA), anti-HA (Sigma-Aldrich, St. Louis, MO), or anti-VP40 antibodies. (Anti-VP40 monoclonal antibody was kindly provided by Ronald Harty, University of Pennsylvania.)

Article Title: BMP4 regulates the hematopoietic stem cell niche
Article Snippet: .. For detection of BMP4 in bone marrow, cell extracts were prepared by sonication in radioimmunoprecipitation assay buffer (50 mM Tris [tris(hydroxymethyl)aminomethane], pH 8; 150 mM NaCl; 0.1% sodium dodecyl sulfate [SDS], 1% nonidet P-40, 0.5% sodium deoxycholate, 1× Roche mini complete protease inhibitor cocktail [Roche]). .. Samples were then incubated on ice for 20 minutes and centrifuged to remove cellular debris.

Article Title: Phosphorylation of VE-cadherin controls endothelial phenotypes via p120-catenin coupling and Rac1 activation
Article Snippet: .. Cells were washed twice with ice-cold PBS and lysed in radioimmunoprecipitation assay buffer containing 50 mM Tris·HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40 (NP-40), 0.5% sodium deoxycholate, 0.1% SDS, and 1× complete protease inhibitor mixture (Roche). .. Total cell lysates were subjected to SDS-PAGE, followed by immunoblotting.

Article Title: A functional genetic screen identifies retinoic acid signaling as a target of histone deacetylase inhibitors
Article Snippet: .. Cells were lysed in radioimmunoprecipitation assay buffer (50 mM Tris, pH 8.0/150 mM NaCl/1% Nonidet P-40/0.5% deoxycholic acid/0.1% SDS) supplemented with protease inhibitors (Complete; Roche, Indianapolis, IN) and 0.2 nM PMSF, and proteins were separated on SDS/10–14% polyacrylamide gels. .. Proteins were transferred to polyvinylidine difluoride membranes (Immobilon P; Millipore, Billerica, MA) and Western blots were probed with the indicated antibodies.

Article Title: DAPK2 is a novel modulator of TRAIL-induced apoptosis
Article Snippet: .. Proteins were extracted using radioimmunoprecipitation assay buffer (RIPA; 50 mM Tris-HCl (pH 7.4), 0.5% (v/v) NP-40, 150 mM NaCl, 1 mM EDTA, 1 mM Na3 VO4 and ‘cOmplete and Mini, EDTA-free protease inhibitor cocktail', the latter used as instructed by the manufacturer; Roche, Mannheim, Germany). ..

Article Title: Loss of the Ubiquitin-conjugating Enzyme UBE2W Results in Susceptibility to Early Postnatal Lethality and Defects in Skin, Immune, and Male Reproductive Systems *
Article Snippet: .. Protein lysates from different organs were generated by lysis in radioimmunoprecipitation assay buffer containing protease inhibitors (Complete Mini, Roche Diagnostics) followed by sonication and centrifugation. .. The supernatants were collected, total protein concentration was determined using the BCA method (Pierce), and supernatants were then stored at −80 °C.

Article Title:
Article Snippet: .. After brief centrifugation, cell pellets were lysed in radioimmunoprecipitation assay buffer containing a commercial protease inhibitor mix (Roche Applied Science, Indianapolis, IN) and phosphatase inhibitor (50 mM sodium fluoride and 10 mM sodium orthovanadate). .. After quantification by Bradford protein assay (Bio-Rad Laboratories, Hercules, CA), the proteins were resolved by 10% SDS-PAGE and transferred onto a nitrocellulose membrane (Bio-Rad Laboratories).

Article Title: The Varicella-Zoster Virus Immediate-Early 63 protein affects chromatin controlled gene transcription in a cell-type dependent manner
Article Snippet: .. Western Blot analysis HeLa or MeWo cells expressing or not wild-type or mutated IE63 were lysed in radioimmunoprecipitation assay buffer (10 mM Tris-HCl pH 8, 100 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% SDS) containing complete protease inhibitor cocktail (Roche), and 5 μg of proteins were separated on a 10% SDS-polyacrylamide gel. ..

Article Title: Proteomic Analyses Reveal Divergent Ubiquitylation Site Patterns in Murine Tissues *
Article Snippet: .. The frozen tissue samples were cryogrinded using a Retsch MM 400 ball mill and lysed in modified radioimmunoprecipitation assay buffer (50 mm Tris pH 7.5, 150 mm NaCl, 1 mm EDTA, 1% Nonidet P-40, 0.1% sodium deoxycholate) supplemented with protease inhibitors (Complete protease inhibitor mixture tablets, Roche Diagnostics) and 5 mm N-ethylmaleimide (Sigma). .. MS Sample Preparation Twenty milligrams of protein from tissue lysates were precipitated in acetone and subsequently re-dissolved in denaturation buffer (6 m urea, 2 m thiourea in 10 mm HEPES pH 8.0).

Article Title: Autophagy Promotes Oligodendrocyte Survival and Function following Dysmyelination in a Long-Lived Myelin Mutant
Article Snippet: .. Protein was extracted using radioimmunoprecipitation assay buffer: 20mM Tris, 150mM NaCl, 0.1% SDS, 1% NP40, and protease inhibitor (Complete, Roche) and quantified using the Quant-iT protein detection kit with the Qubit flourometer according to manufacturer’s protocols (Invitrogen). .. A total of 20 µg of each sample was run on a 12% acrylamide gel and blotted onto Immobilon-P membranes (Millipore) using the Mini-PROTEAN electrophoresis system (Bio-Rad).

Article Title: The Zebra fish cassiopeia Mutant Reveals that SIL Is Required for Mitotic Spindle Organization ▿ Mutant Reveals that SIL Is Required for Mitotic Spindle Organization ▿ §
Article Snippet: .. Protein lysates were extracted from 24- to 30-hpf zebrafish embryos with radioimmunoprecipitation assay buffer supplemented with complete protease inhibitor tablets (Roche). .. Forty micrograms of total protein was subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose for immunoblotting with polyclonal γ-tubulin (Sigma).

Article Title: Hypoxia and Glucocorticoid Signaling Converge to Regulate Macrophage Migration Inhibitory Factor Gene Expression
Article Snippet: .. For whole cell lysates, cells were harvested and lysed in radioimmunoprecipitation assay buffer (100 m M Tris [pH 7.4], 150 m M NaCl, 1% Nonidet P40, 2.5% sodium deoxycholate, and 1 m M EDTA) with Complete protease inhibitor (Roche, Indianapolis, IN). .. Cell lysates and nuclear extracts were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed with one of the following antibodies: anti-GR α (1:1,000 dilution) (GR α -clone 41; BD Biosciences, San Jose, CA) anti-phosphoGR (1:2,000; New England Biolabs, Ipswich, MA), anti–ATF-1 (1:2,000; Santa Cruz Biotechnology, Santa Cruz, CA), anti-CREB (1:2,000; Santa Cruz Biotechnology), anti HIF-1 α (1:2,000; Santa Cruz Biotechnology), and anti-tubulin (1:2,000; Sigma, St. Louis, MO).

Article Title: Phosphorylation of the Herpes Simplex Virus Tegument Protein VP22 Has No Effect on Incorporation of VP22 into the Virus but Is Involved in Optimal Expression and Virion Packaging of ICP0
Article Snippet: .. Radioactive labeling was conducted by the addition of 100 μCi of [32 P]orthophosphate (Amersham) per dish, and the cells were incubated at 37°C for a further 13 h. After washing off the unincorporated label with PBS, proteins were extracted with 1 ml of radioimmunoprecipitation assay buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.1% SDS, 1% Na deoxycholate, 0.5% Triton X-100) containing protease inhibitors (Miniprotease inhibitors; Roche Diagnostics Ltd.). .. Proteins from the soluble fraction were immunoprecipitated with the anti-VP22 polyclonal antibody AGV031, and after extensive washing of the protein A Sepharose beads (Amersham), proteins were analyzed by SDS-PAGE and autoradiography.

Article Title: HIV Vpu Interferes with NF-κB Activity but Not with Interferon Regulatory Factor 3
Article Snippet: .. Cells were lysed in radioimmunoprecipitation assay buffer supplemented with complete protease inhibitor (Roche). .. Proteins were separated by SDS-PAGE (Invitrogen) and transferred to polyvinylidene difluoride membranes (Pierce).

Article Title: The Zebra fish cassiopeia Mutant Reveals that SIL Is Required for Mitotic Spindle Organization ▿ Mutant Reveals that SIL Is Required for Mitotic Spindle Organization ▿ §
Article Snippet: .. For immunoblotting, HEK293T cells were lysed with radioimmunoprecipitation assay buffer supplemented with complete protease inhibitors (Roche). .. Thirty micrograms of total protein was subjected to 6% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose.

Immunoprecipitation:

Article Title: Phosphorylation of VE-cadherin controls endothelial phenotypes via p120-catenin coupling and Rac1 activation
Article Snippet: Paragraph title: Western blot analysis and immunoprecipitation. ... Cells were washed twice with ice-cold PBS and lysed in radioimmunoprecipitation assay buffer containing 50 mM Tris·HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40 (NP-40), 0.5% sodium deoxycholate, 0.1% SDS, and 1× complete protease inhibitor mixture (Roche).

Article Title: Phosphorylation of the Herpes Simplex Virus Tegument Protein VP22 Has No Effect on Incorporation of VP22 into the Virus but Is Involved in Optimal Expression and Virion Packaging of ICP0
Article Snippet: Radioactive labeling was conducted by the addition of 100 μCi of [32 P]orthophosphate (Amersham) per dish, and the cells were incubated at 37°C for a further 13 h. After washing off the unincorporated label with PBS, proteins were extracted with 1 ml of radioimmunoprecipitation assay buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.1% SDS, 1% Na deoxycholate, 0.5% Triton X-100) containing protease inhibitors (Miniprotease inhibitors; Roche Diagnostics Ltd.). .. Proteins from the soluble fraction were immunoprecipitated with the anti-VP22 polyclonal antibody AGV031, and after extensive washing of the protein A Sepharose beads (Amersham), proteins were analyzed by SDS-PAGE and autoradiography.

Lysis:

Article Title: Phosphorylation of VE-cadherin controls endothelial phenotypes via p120-catenin coupling and Rac1 activation
Article Snippet: Cells were washed twice with ice-cold PBS and lysed in radioimmunoprecipitation assay buffer containing 50 mM Tris·HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40 (NP-40), 0.5% sodium deoxycholate, 0.1% SDS, and 1× complete protease inhibitor mixture (Roche). .. For immunoprecipitation assay, total cell lysates were harvested with HEPES lysis buffer with NP-40 containing 50 mM HEPES (pH 7.4), 150 mM NaCl, 1% NP-40, 5 mM EDTA, and 1× complete protease inhibitor mixture (Roche) and incubated with the appropriate antibody and Dynabeads Protein G (Invitrogen).

Article Title: Loss of the Ubiquitin-conjugating Enzyme UBE2W Results in Susceptibility to Early Postnatal Lethality and Defects in Skin, Immune, and Male Reproductive Systems *
Article Snippet: .. Protein lysates from different organs were generated by lysis in radioimmunoprecipitation assay buffer containing protease inhibitors (Complete Mini, Roche Diagnostics) followed by sonication and centrifugation. .. The supernatants were collected, total protein concentration was determined using the BCA method (Pierce), and supernatants were then stored at −80 °C.

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  • 92
    Roche radioimmunoprecipitation buffer ripa
    Immunoblot analysis of Aβ species from <t>RIPA</t> soluble lysates from human subjects . Human hippocampi from Alzheimer's disease (AD), pathological aging (PA) and normal (N) cohorts were extracted with RIPA. Representative anti-82E1 immunoblot of RIPA extracted lysates of Alzheimer's disease (AD), pathologic aging (PA) and control (N) subjects with ( A ) and without ( B ) a post-mortem clinical pathological diagnosis of cerebral amyloid angiopathy (+CAA). Longer exposure panels below highlight no significant differences between the three cohorts in the dimer/trimer molecular weight range. Aβ, amyloid β; RIPA, <t>radioimmunoprecipitation</t> buffer.
    Radioimmunoprecipitation Buffer Ripa, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/radioimmunoprecipitation buffer ripa/product/Roche
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    radioimmunoprecipitation buffer ripa - by Bioz Stars, 2020-04
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    93
    Roche rip buffer
    p68 interacting single exonic lncRNAs. (A) We filtered 54 single exonic lncRNA transcripts from 72 single exonic lncRNA transcripts of <t>RIP-seq,</t> transcripts with more than 80% of similarity with its genomic region has been selected and plotted based on their Log2 fold enrichment ratio and few transcripts were selected (red) for further validation by RIP-PCR in <t>HEK293T</t> cells. (B) RIP-PCR of selected transcripts in p68, SF2 (negative control) and RPL7 (negative control) antibody pulldown complexes.
    Rip Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 93/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rip buffer/product/Roche
    Average 93 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    rip buffer - by Bioz Stars, 2020-04
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    96
    Roche rip lysis buffer
    C ircBIRC6 interacts with and sequester miR-34a and miR-145. a RT-qPCR analysis of cytoplasmic-to-nuclear expression ratios of circBIRC6 , circCORO1C , CDR1as , GAPDH , and U6 in <t>hESCs.</t> b <t>RIP</t> analysis of circBIRC6 , linear BIRC6 (linB) , circCORO1C
    Rip Lysis Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 96/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rip lysis buffer/product/Roche
    Average 96 stars, based on 8 article reviews
    Price from $9.99 to $1999.99
    rip lysis buffer - by Bioz Stars, 2020-04
    96/100 stars
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    Immunoblot analysis of Aβ species from RIPA soluble lysates from human subjects . Human hippocampi from Alzheimer's disease (AD), pathological aging (PA) and normal (N) cohorts were extracted with RIPA. Representative anti-82E1 immunoblot of RIPA extracted lysates of Alzheimer's disease (AD), pathologic aging (PA) and control (N) subjects with ( A ) and without ( B ) a post-mortem clinical pathological diagnosis of cerebral amyloid angiopathy (+CAA). Longer exposure panels below highlight no significant differences between the three cohorts in the dimer/trimer molecular weight range. Aβ, amyloid β; RIPA, radioimmunoprecipitation buffer.

    Journal: Alzheimer's Research & Therapy

    Article Title: Overlapping profiles of A? peptides in the Alzheimer's disease and pathological aging brains

    doi: 10.1186/alzrt121

    Figure Lengend Snippet: Immunoblot analysis of Aβ species from RIPA soluble lysates from human subjects . Human hippocampi from Alzheimer's disease (AD), pathological aging (PA) and normal (N) cohorts were extracted with RIPA. Representative anti-82E1 immunoblot of RIPA extracted lysates of Alzheimer's disease (AD), pathologic aging (PA) and control (N) subjects with ( A ) and without ( B ) a post-mortem clinical pathological diagnosis of cerebral amyloid angiopathy (+CAA). Longer exposure panels below highlight no significant differences between the three cohorts in the dimer/trimer molecular weight range. Aβ, amyloid β; RIPA, radioimmunoprecipitation buffer.

    Article Snippet: Briefly, for ELISA and IP/MS, the cryo-pulverized tissue was sequentially extracted with Tris-buffered saline (TBS), radioimmunoprecipitation buffer (RIPA), 2% sodium dodecyl sulfate (SDS) and 70% formic acid (FA) containing protease inhibitor cocktail (Roche, Indianapolis, IN, USA) as described before at a concentration of 300 mg/mL [ ].

    Techniques: Cellular Antioxidant Activity Assay, Molecular Weight

    p68 interacting single exonic lncRNAs. (A) We filtered 54 single exonic lncRNA transcripts from 72 single exonic lncRNA transcripts of RIP-seq, transcripts with more than 80% of similarity with its genomic region has been selected and plotted based on their Log2 fold enrichment ratio and few transcripts were selected (red) for further validation by RIP-PCR in HEK293T cells. (B) RIP-PCR of selected transcripts in p68, SF2 (negative control) and RPL7 (negative control) antibody pulldown complexes.

    Journal: RNA Biology

    Article Title: DDX5/p68 associated lncRNA LOC284454 is differentially expressed in human cancers and modulates gene expression

    doi: 10.1080/15476286.2017.1397261

    Figure Lengend Snippet: p68 interacting single exonic lncRNAs. (A) We filtered 54 single exonic lncRNA transcripts from 72 single exonic lncRNA transcripts of RIP-seq, transcripts with more than 80% of similarity with its genomic region has been selected and plotted based on their Log2 fold enrichment ratio and few transcripts were selected (red) for further validation by RIP-PCR in HEK293T cells. (B) RIP-PCR of selected transcripts in p68, SF2 (negative control) and RPL7 (negative control) antibody pulldown complexes.

    Article Snippet: Briefly, HEK293T cells were lysed in RIP buffer containing 0.5% Nonidet P-40, 150 mM NaCl, 2 mM MgCl2 , 1 mM DTT, 0.1 mM EDTA, 10% glycerol, 5 mM vanadyl ribonucleoside complex (VRC), 100 U/ml RNase inhibitor and 1X protease inhibitor cocktail (Roche).

    Techniques: Polymerase Chain Reaction, Negative Control

    RIP-seq strategy and data analysis. (A) Schematic representation of the domain structure of p68 protein. (B) Outline of RIP-seq analysis pipeline. (C) RIP in HEK293T cells using p68 antibody (top panel). Specificity of the pull down was ascertained by enrichment of SRA RNA in p68 IP (bottom panel). Input represents 10% of the total sample. (D) Scatter plot showing the pattern of enriched transcripts. Read counts (log 2) for each transcript are plotted against that of control (Control+2 and IP+2 to avoid division by zero). Reads with read coverage > 10 are plotted. (E) Different types of transcripts obtained in the RIP. SE – single exonic lncRNA transcripts and ME – Multi exonic lncRNA transcripts.

    Journal: RNA Biology

    Article Title: DDX5/p68 associated lncRNA LOC284454 is differentially expressed in human cancers and modulates gene expression

    doi: 10.1080/15476286.2017.1397261

    Figure Lengend Snippet: RIP-seq strategy and data analysis. (A) Schematic representation of the domain structure of p68 protein. (B) Outline of RIP-seq analysis pipeline. (C) RIP in HEK293T cells using p68 antibody (top panel). Specificity of the pull down was ascertained by enrichment of SRA RNA in p68 IP (bottom panel). Input represents 10% of the total sample. (D) Scatter plot showing the pattern of enriched transcripts. Read counts (log 2) for each transcript are plotted against that of control (Control+2 and IP+2 to avoid division by zero). Reads with read coverage > 10 are plotted. (E) Different types of transcripts obtained in the RIP. SE – single exonic lncRNA transcripts and ME – Multi exonic lncRNA transcripts.

    Article Snippet: Briefly, HEK293T cells were lysed in RIP buffer containing 0.5% Nonidet P-40, 150 mM NaCl, 2 mM MgCl2 , 1 mM DTT, 0.1 mM EDTA, 10% glycerol, 5 mM vanadyl ribonucleoside complex (VRC), 100 U/ml RNase inhibitor and 1X protease inhibitor cocktail (Roche).

    Techniques:

    LOC284454 RNA is a stable RNA that interacts with p68 in vivo. (A) LOC284454 is located on human chromosome 19 (chr19:13945330-13947473). Genomic sequence analysis showed that the RNA is 2 nucleotide downstream to the 3′ end of miR-23a∼27a∼24-2 cluster. The transcription start site and the validated promoter region are marked. (B) Schematic representation of LOC284454 primary transcript. The Drosha cleavage site which generates the miRNA cluster and the LOC284454 is marked. The blue arrow depicts the primer annealing positions on the primary transcript and black arrows on the LOC284454 specific region. (C) Drosha silencing was carried out with pool of 2 independent siRNAs. A representative western blot using anti Drosha antibody shows reduced Drosha level at 72 h post transfection. (D) The 2.2 kb full length PCR product was detected from genomic DNA (lane 2) and Drosha silenced cDNA (lane 4) but not from Drosha scrambled siRNA cDNA template (lane 3). The 1.77 kb LOC284454 was detected from both Drosha control cDNA (lane 6) and Drosha silenced cDNA (lane 7). Genomic DNA was used in lane 2 and lane 5 as positive control for PCR. (E) In vivo pull-down of LOC284454 in HEK293T cells transfected with plasmid containing LOC284454 fused with S1 aptamer at the 3' end. Presence of p68 was observed in the LOC284454 -S1 aptamer pull-down but not in the mock pull-down. (F) RNA immunoprecipitation with p68 antibody shows the presence of p68 (top) and enrichment of LOC284454 in the IP fraction (bottom). (G) LOC284454 RNA enrichment in IP fractions of anti-p68 antibody, anti-SF2 antibody, anti-RPL7 antibody and Rabbit anti-IgG, *** – p ≤0.0001 and NS-non-significant paired two tailed t test (H) RIP-PCR from p68 IP fraction for primary transcript (2.2kb), LOC284454 (1.7kb) and miRNA cluster, SRA lncRNA was used as a positive control for p68 specificity. (I) RNA stability assay of LOC284454 RNA and 18S rRNA in the presence of actinomycin D (10µg/ml). These data show the means ± SD from three independent experiments in triplicate.

    Journal: RNA Biology

    Article Title: DDX5/p68 associated lncRNA LOC284454 is differentially expressed in human cancers and modulates gene expression

    doi: 10.1080/15476286.2017.1397261

    Figure Lengend Snippet: LOC284454 RNA is a stable RNA that interacts with p68 in vivo. (A) LOC284454 is located on human chromosome 19 (chr19:13945330-13947473). Genomic sequence analysis showed that the RNA is 2 nucleotide downstream to the 3′ end of miR-23a∼27a∼24-2 cluster. The transcription start site and the validated promoter region are marked. (B) Schematic representation of LOC284454 primary transcript. The Drosha cleavage site which generates the miRNA cluster and the LOC284454 is marked. The blue arrow depicts the primer annealing positions on the primary transcript and black arrows on the LOC284454 specific region. (C) Drosha silencing was carried out with pool of 2 independent siRNAs. A representative western blot using anti Drosha antibody shows reduced Drosha level at 72 h post transfection. (D) The 2.2 kb full length PCR product was detected from genomic DNA (lane 2) and Drosha silenced cDNA (lane 4) but not from Drosha scrambled siRNA cDNA template (lane 3). The 1.77 kb LOC284454 was detected from both Drosha control cDNA (lane 6) and Drosha silenced cDNA (lane 7). Genomic DNA was used in lane 2 and lane 5 as positive control for PCR. (E) In vivo pull-down of LOC284454 in HEK293T cells transfected with plasmid containing LOC284454 fused with S1 aptamer at the 3' end. Presence of p68 was observed in the LOC284454 -S1 aptamer pull-down but not in the mock pull-down. (F) RNA immunoprecipitation with p68 antibody shows the presence of p68 (top) and enrichment of LOC284454 in the IP fraction (bottom). (G) LOC284454 RNA enrichment in IP fractions of anti-p68 antibody, anti-SF2 antibody, anti-RPL7 antibody and Rabbit anti-IgG, *** – p ≤0.0001 and NS-non-significant paired two tailed t test (H) RIP-PCR from p68 IP fraction for primary transcript (2.2kb), LOC284454 (1.7kb) and miRNA cluster, SRA lncRNA was used as a positive control for p68 specificity. (I) RNA stability assay of LOC284454 RNA and 18S rRNA in the presence of actinomycin D (10µg/ml). These data show the means ± SD from three independent experiments in triplicate.

    Article Snippet: Briefly, HEK293T cells were lysed in RIP buffer containing 0.5% Nonidet P-40, 150 mM NaCl, 2 mM MgCl2 , 1 mM DTT, 0.1 mM EDTA, 10% glycerol, 5 mM vanadyl ribonucleoside complex (VRC), 100 U/ml RNase inhibitor and 1X protease inhibitor cocktail (Roche).

    Techniques: In Vivo, Sequencing, Western Blot, Transfection, Polymerase Chain Reaction, Positive Control, Plasmid Preparation, Immunoprecipitation, Two Tailed Test, Stability Assay

    C ircBIRC6 interacts with and sequester miR-34a and miR-145. a RT-qPCR analysis of cytoplasmic-to-nuclear expression ratios of circBIRC6 , circCORO1C , CDR1as , GAPDH , and U6 in hESCs. b RIP analysis of circBIRC6 , linear BIRC6 (linB) , circCORO1C

    Journal: Nature Communications

    Article Title: The circular RNA circBIRC6 participates in the molecular circuitry controlling human pluripotency

    doi: 10.1038/s41467-017-01216-w

    Figure Lengend Snippet: C ircBIRC6 interacts with and sequester miR-34a and miR-145. a RT-qPCR analysis of cytoplasmic-to-nuclear expression ratios of circBIRC6 , circCORO1C , CDR1as , GAPDH , and U6 in hESCs. b RIP analysis of circBIRC6 , linear BIRC6 (linB) , circCORO1C

    Article Snippet: In brief, 2 × 107 H9 hESCs were UV-crosslinked at 600 mj cm−2 and lysed in 100 μl RIP lysis buffer containing a proteinase inhibitor cocktail (Roche) and RNase inhibitor (Promega).

    Techniques: Quantitative RT-PCR, Expressing

    C ircBIRC6 interacts with and sequester miR-34a and miR-145. a RT-qPCR analysis of cytoplasmic-to-nuclear expression ratios of circBIRC6 , circCORO1C , CDR1as , GAPDH , and U6 in hESCs. b RIP analysis of circBIRC6 , linear BIRC6 (linB) , circCORO1C , linear CORO1C (LinC) , and CDR1as in hESCs using antibodies against AGO2. Immunoblotting (IB) of immunoprecipitated (IP) AGO2 protein is shown. The RIP enrichment of the AGO2-associated circular RNAs (as indicated) was measured by RT-qPCR, and each value was normalized to the level of input RNA used in RIP analysis. c RT-qPCR analysis of circBIRC6 or circCORO1C pulled-down by biotinylated miR-34a or miR-145 mimics in hESCs. d Schematic illustration showing the 3′ UTR of luciferase reporters containing the complete circBIRC6 sequence (luc-circB) or circBIRC6 sequence with deletions of miR-34a (luc-circBm1 and luc-circBm2) or miR-145 (luc-circBm3 and luc-circBm4) binding sites. e , f Reporter assays showing the luciferase activity of luc-circB and luc-circBm1-m4 in 293T cells co-transfected with e miR-34a or f miR-145 mimics, or a scrambled oligonucleotide (control). g RT-qPCR analysis of circBIRC6 , miR-34a , and miR-145 expression during hESC differentiation. IVD: in vitro differentiation day. h RT-qPCR and i immunoblot analyses of the indicated gene and protein expression in hESCs transfected with miR-34a or miR-145 mimics, or scrambled oligonucleotide 3 days post transfection. Protein level quantification was normalized to actin and shown below each panel. Quantitative data from three independent experiments is presented as mean ± SD (error bars). P -values were determined by two-tailed two-sample t -tests (* P

    Journal: Nature Communications

    Article Title: The circular RNA circBIRC6 participates in the molecular circuitry controlling human pluripotency

    doi: 10.1038/s41467-017-01216-w

    Figure Lengend Snippet: C ircBIRC6 interacts with and sequester miR-34a and miR-145. a RT-qPCR analysis of cytoplasmic-to-nuclear expression ratios of circBIRC6 , circCORO1C , CDR1as , GAPDH , and U6 in hESCs. b RIP analysis of circBIRC6 , linear BIRC6 (linB) , circCORO1C , linear CORO1C (LinC) , and CDR1as in hESCs using antibodies against AGO2. Immunoblotting (IB) of immunoprecipitated (IP) AGO2 protein is shown. The RIP enrichment of the AGO2-associated circular RNAs (as indicated) was measured by RT-qPCR, and each value was normalized to the level of input RNA used in RIP analysis. c RT-qPCR analysis of circBIRC6 or circCORO1C pulled-down by biotinylated miR-34a or miR-145 mimics in hESCs. d Schematic illustration showing the 3′ UTR of luciferase reporters containing the complete circBIRC6 sequence (luc-circB) or circBIRC6 sequence with deletions of miR-34a (luc-circBm1 and luc-circBm2) or miR-145 (luc-circBm3 and luc-circBm4) binding sites. e , f Reporter assays showing the luciferase activity of luc-circB and luc-circBm1-m4 in 293T cells co-transfected with e miR-34a or f miR-145 mimics, or a scrambled oligonucleotide (control). g RT-qPCR analysis of circBIRC6 , miR-34a , and miR-145 expression during hESC differentiation. IVD: in vitro differentiation day. h RT-qPCR and i immunoblot analyses of the indicated gene and protein expression in hESCs transfected with miR-34a or miR-145 mimics, or scrambled oligonucleotide 3 days post transfection. Protein level quantification was normalized to actin and shown below each panel. Quantitative data from three independent experiments is presented as mean ± SD (error bars). P -values were determined by two-tailed two-sample t -tests (* P

    Article Snippet: In brief, 2 × 107 H9 hESCs were UV-crosslinked at 600 mj cm−2 and lysed in 100 μl RIP lysis buffer containing a proteinase inhibitor cocktail (Roche) and RNase inhibitor (Promega).

    Techniques: Quantitative RT-PCR, Expressing, Immunoprecipitation, Luciferase, Sequencing, Binding Assay, Activity Assay, Transfection, In Vitro, Two Tailed Test

    ESRP1 promotes the biogenesis of circ BIRC6 in hESCs. a RT-qPCR analysis of the splicing factors (as indicated) in H9 hESCs. b RT-qPCR analysis of the expression of circBIRC6 (exon 9–2), linear BIRC6 (linBIRC6 exon 9–10 and exon 54–55), NANOG , and OCT4 in ESRP1-knockdown hESCs (shESRP-1 and shESRP-2). c Schematic illustrating the putative ESRP1-binding sites on the flanking introns in the circB-s minigene. The 5′ terminus of the circular exons of circBIRC6 was defined as position 0. Putative ESRP1-binding sites A, B, and C are located in the intron at the 5′ terminus of the circBIRC6 exon (position: −1282 to −1287), within the circBIRC6 exon (position: 84–89), and on the intron at the 3′ terminus of the circBIRC6 exon (position: 1733–1738). d RIP analysis of ESRP1-binding to circB-s and circB-s-Em minigenes in hESCs. Bound complexes were pulled-down using an antibody against ESRP1. RT-qPCR was then used to measure circB-s binding to ESRP1. Values were normalized to the level of background RIP, as detected by an IgG isotype control. Inserted panel: Immunoblotting (IB) of immunoprecipitated (IP) ESRP1 protein. e RT-qPCR analysis of the expression of circBIRC6 relative to GAPDH in hESCs. Cells were co-transfected with shRNA that target ESRP1 and a circBIRC6 minigene (circB-s), or circBIRC6 minigene containing deleted ESRP1-binding sites (circB-s-Em). f Schematic showing sites of insertion of ESRP1-binding sequence and the location of primers used for detecting the two minigenes (EGFR and ZEB1). g RT-PCR analysis using circRNA-specific or precursor specific primers on RNA from hESCs that were transfected with the indicated minigenes, with or without shRNA-mediated disruption of ESRP1. Quantitative data from three independent experiments is presented as mean ± SD (error bars). P -values were determined by two-tailed two-sample t -tests (* P

    Journal: Nature Communications

    Article Title: The circular RNA circBIRC6 participates in the molecular circuitry controlling human pluripotency

    doi: 10.1038/s41467-017-01216-w

    Figure Lengend Snippet: ESRP1 promotes the biogenesis of circ BIRC6 in hESCs. a RT-qPCR analysis of the splicing factors (as indicated) in H9 hESCs. b RT-qPCR analysis of the expression of circBIRC6 (exon 9–2), linear BIRC6 (linBIRC6 exon 9–10 and exon 54–55), NANOG , and OCT4 in ESRP1-knockdown hESCs (shESRP-1 and shESRP-2). c Schematic illustrating the putative ESRP1-binding sites on the flanking introns in the circB-s minigene. The 5′ terminus of the circular exons of circBIRC6 was defined as position 0. Putative ESRP1-binding sites A, B, and C are located in the intron at the 5′ terminus of the circBIRC6 exon (position: −1282 to −1287), within the circBIRC6 exon (position: 84–89), and on the intron at the 3′ terminus of the circBIRC6 exon (position: 1733–1738). d RIP analysis of ESRP1-binding to circB-s and circB-s-Em minigenes in hESCs. Bound complexes were pulled-down using an antibody against ESRP1. RT-qPCR was then used to measure circB-s binding to ESRP1. Values were normalized to the level of background RIP, as detected by an IgG isotype control. Inserted panel: Immunoblotting (IB) of immunoprecipitated (IP) ESRP1 protein. e RT-qPCR analysis of the expression of circBIRC6 relative to GAPDH in hESCs. Cells were co-transfected with shRNA that target ESRP1 and a circBIRC6 minigene (circB-s), or circBIRC6 minigene containing deleted ESRP1-binding sites (circB-s-Em). f Schematic showing sites of insertion of ESRP1-binding sequence and the location of primers used for detecting the two minigenes (EGFR and ZEB1). g RT-PCR analysis using circRNA-specific or precursor specific primers on RNA from hESCs that were transfected with the indicated minigenes, with or without shRNA-mediated disruption of ESRP1. Quantitative data from three independent experiments is presented as mean ± SD (error bars). P -values were determined by two-tailed two-sample t -tests (* P

    Article Snippet: In brief, 2 × 107 H9 hESCs were UV-crosslinked at 600 mj cm−2 and lysed in 100 μl RIP lysis buffer containing a proteinase inhibitor cocktail (Roche) and RNase inhibitor (Promega).

    Techniques: Quantitative RT-PCR, Expressing, Binding Assay, Immunoprecipitation, Transfection, shRNA, Sequencing, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test