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Roche ethylenediaminetetraacetic acid
Ethylenediaminetetraacetic Acid, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 99 stars, based on 7 article reviews
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ethylenediaminetetraacetic acid - by Bioz Stars, 2020-04
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Transduction:

Article Title: Activation of MAPK/ERK signaling by Burkholderia pseudomallei cycle inhibiting factor (Cif)
Article Snippet: The composition of the lysis buffer was as follows: 25 mM Tris-HCl (pH 7.5), 100 mM NaCl, 2.5 mM EDTA, 2.5 mM EGTA, 20 mM NaF, 1 mM Na3 VO4 , 20 mM Sodium β-Glycerophosphate, 10 mM Sodium Pyrophosphate, 0.5% Triton X-100, Roche protease inhibitor cocktail and 0.1% β-Mercaptoethanol. .. The composition of the lysis buffer was as follows: 25 mM Tris-HCl (pH 7.5), 100 mM NaCl, 2.5 mM EDTA, 2.5 mM EGTA, 20 mM NaF, 1 mM Na3 VO4 , 20 mM Sodium β-Glycerophosphate, 10 mM Sodium Pyrophosphate, 0.5% Triton X-100, Roche protease inhibitor cocktail and 0.1% β-Mercaptoethanol.

Clone Assay:

Article Title: Crystal Structure of Penicillin-Binding Protein 3 (PBP3) from Escherichia coli
Article Snippet: The PCR fragment was cloned into the pJet1.2/blunt cloning vector (Fermentas), sequenced, digested with XmaI and BamHI and inserted into the same sites of plasmid pET-52b(+). .. The strep-tag- PBP388–165 was isolated from E. coli C41(DE3) harbouring pDML2042 grown at 37°C in 2XYT medium in the presence of 0.5 mM IPTG for 3 h. The harvested cells were suspended in 40 ml of 100 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA containing a protease inhibitor cocktail (Roche) (buffer C), broken 5 times into a high-pressure homogenizer (Emulsiflex-C3 Avestin Inc.) and centrifuged at 25000 g for 40 min.

Centrifugation:

Article Title: Centriolar Satellites Control GABARAP Ubiquitination and GABARAP-Mediated Autophagy
Article Snippet: Ubiquitination mass spectrometry Cells were lysed in ice-cold buffer (Tris-HCl 20 mM pH6.8, 0.5% (w/v) Triton X-100, 150 mM NaCl, 5 mM EDTA, phosphatase inhibitor cocktail and mammalian protease inhibitor cocktail [Roche], and 20 mM N-Ethylmaleimide). .. The lysates were then clarified by centrifugation (5 min, full speed, 4°C) and the supernatants containing GFP-GABARAP ubiquitinated conjugates were subjected to immunoprecipitation at 4°C with GFP-TRAP beads for 2 hr.

Article Title: The Structure and Regulation of Human Muscle α-Actinin
Article Snippet: .. After induction, cells were harvested by centrifugation and the cell pellet from one liter of high-density shaking cultures (OD600 ∼5 - 8 A.U.) were re-suspended in 50 ml of lysis buffer 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5mM EDTA, 0.1% Triton X-100 supplemented with antiproteases (Complete™ Protease Inhibitor Cocktail Tablets, Roche). ..

Article Title: DNA Methylation Analysis in the Intestinal Epithelium--Effect of Cell Separation on Gene Expression and Methylation Profile
Article Snippet: Subsequent purification of IEC from accompanying mononuclear and stromal cells may be accomplished by density centrifugation or cell sorting using immunomagnetic beads or flow cytometry. .. Four pooled biopsy samples were then subjected to either chemical digestion using a combination of EDTA and DTT, or enzymatic digestion with Liberase (a blend of collagenase I/II and a neutral protease; Roche Applied Science) and hyaluronidase.

Amplification:

Article Title: Maintaining mRNA Integrity during Decalcification of Mineralized Tissues
Article Snippet: Quantitative PCR RNA isolated from EDTA or RNAlater /EDTA decalcified whole tibiae (500 ng total RNA) was reverse-transcribed as per the manufacturer’s protocol in a 20µL reaction using the Transcriptor High Fidelity cDNA Synthesis Kit (Roche, Germany). .. Cycling conditions for qPCR reactions were; Pre-incubation, 1 cycle of 95°C (5 minutes, 4.8°C/s ramp rate); Amplification, 45 cycles of 95°C (10 seconds 4.8°C/s ramp rate), 60°C (30 seconds 2.5°C/s ramp rate), 72°C (10 seconds, 4.8°C/s ramp rate); Melting Curve, 1 cycle of 95°C (5 seconds 4.8°C/s ramp rate, 65°C (1 minute 2.5°C/s ramp rate), 97°C (0.11°C/s ramp rate).

Article Title: Crystal Structure of Penicillin-Binding Protein 3 (PBP3) from Escherichia coli
Article Snippet: PBP388–165 : Production, Purification, Crystallization, Data Collection and Structure Refinement The ftsI fragment encoding PBP388–165 was amplified by PCR using plasmid pMVRI as template and oligonucleotides 5′-GGA CCCGGG GTAAAAGCGATTTGGGCTGACCC-3′ and 5′-GCC GGATCC TTAAGAC TCTTCACGCAGATGAATCCC-3′ as primers (XmaI and BamHI are underlined). .. The strep-tag- PBP388–165 was isolated from E. coli C41(DE3) harbouring pDML2042 grown at 37°C in 2XYT medium in the presence of 0.5 mM IPTG for 3 h. The harvested cells were suspended in 40 ml of 100 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA containing a protease inhibitor cocktail (Roche) (buffer C), broken 5 times into a high-pressure homogenizer (Emulsiflex-C3 Avestin Inc.) and centrifuged at 25000 g for 40 min.

Expressing:

Article Title: Effective Formation of the Segregation-Competent Complex Determines Successful Partitioning of the Bovine Papillomavirus Genome during Cell Division ▿Effective Formation of the Segregation-Competent Complex Determines Successful Partitioning of the Bovine Papillomavirus Genome during Cell Division ▿ †
Article Snippet: .. Briefly, COS-7 cell extracts were transfected with 1 μg expression plasmid pCG and lysed in 100 μl of lysis buffer (50 mM Tris-HCl [pH 7.5], 100 mM KCl, 0.1 mM EDTA, 0.35% Nonidet P-40, 10 mM dithiothreitol [DTT], and protease inhibitors [Roche]) on ice for 30 min. For gel shift assays, 0.2 μl of cell extract was incubated in 10 μl binding buffer (10 mM Tris-HCl [pH 7.5], 100 mM KCl, 2 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride [PMSF], 15% glycerol, 5 mg of bovine serum albumin [BSA] per ml, protease inhibitors [Roche]) at room temperature for 20 min in the presence of 1 μg of sonicated salmon sperm DNA and 0.5 ng of a 32 P-labeled, double-stranded, high-affinity probe for BPV1 E2 binding site 9 (BS9; 5′-ACAAAGT ACCGTTGCCGGT CGAA-3′ [binding site is shown in bold]). ..

Article Title: Proteolytic cleavage and truncation of NDRG1 in human prostate cancer cells, but not normal prostate epithelial cells
Article Snippet: PC3MM/Tet-Flag-Drg-1 selected cells were treated with 1.1 μg/ml tetracycline in RPMI 1640 for 24 h to induce NDRG1 expression. .. Cells were then lysed using RIPA lysis buffer [65 mM Tris/HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1% (v/v) Triton X-100, 0.1% (w/v) SDS, 0.5% (w/v) sodium deoxycholate, 10% (v/v) glycerol, protease inhibitor cocktail [Complete™ EDTA-free; Roche Applied Science] and PhosStop and sonicated on ice.

Article Title: The Structure and Regulation of Human Muscle α-Actinin
Article Snippet: Paragraph title: Protein Expression and Purification for Biochemical Studies ... After induction, cells were harvested by centrifugation and the cell pellet from one liter of high-density shaking cultures (OD600 ∼5 - 8 A.U.) were re-suspended in 50 ml of lysis buffer 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5mM EDTA, 0.1% Triton X-100 supplemented with antiproteases (Complete™ Protease Inhibitor Cocktail Tablets, Roche).

Cytometry:

Article Title: DNA Methylation Analysis in the Intestinal Epithelium--Effect of Cell Separation on Gene Expression and Methylation Profile
Article Snippet: Subsequent purification of IEC from accompanying mononuclear and stromal cells may be accomplished by density centrifugation or cell sorting using immunomagnetic beads or flow cytometry. .. Four pooled biopsy samples were then subjected to either chemical digestion using a combination of EDTA and DTT, or enzymatic digestion with Liberase (a blend of collagenase I/II and a neutral protease; Roche Applied Science) and hyaluronidase.

Construct:

Article Title: The Structure and Regulation of Human Muscle α-Actinin
Article Snippet: The plasmids were transfected into suitable E. coli host strains for high-level expression that were defined based on protein expression optimization screened for each constructs. .. After induction, cells were harvested by centrifugation and the cell pellet from one liter of high-density shaking cultures (OD600 ∼5 - 8 A.U.) were re-suspended in 50 ml of lysis buffer 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5mM EDTA, 0.1% Triton X-100 supplemented with antiproteases (Complete™ Protease Inhibitor Cocktail Tablets, Roche).

SYBR Green Assay:

Article Title: Maintaining mRNA Integrity during Decalcification of Mineralized Tissues
Article Snippet: Quantitative PCR RNA isolated from EDTA or RNAlater /EDTA decalcified whole tibiae (500 ng total RNA) was reverse-transcribed as per the manufacturer’s protocol in a 20µL reaction using the Transcriptor High Fidelity cDNA Synthesis Kit (Roche, Germany). .. Quantitative PCR (qPCR) reactions containing 2.5 ng cDNA were prepared to 5µL in 384 well PCR plates (Roche) using the LightCycler® 480 SYBR Green I Master (Roche) as per the manufacturer’s instructions and run on a LC480 II thermo cycler (Roche).

Incubation:

Article Title: Effective Formation of the Segregation-Competent Complex Determines Successful Partitioning of the Bovine Papillomavirus Genome during Cell Division ▿Effective Formation of the Segregation-Competent Complex Determines Successful Partitioning of the Bovine Papillomavirus Genome during Cell Division ▿ †
Article Snippet: .. Briefly, COS-7 cell extracts were transfected with 1 μg expression plasmid pCG and lysed in 100 μl of lysis buffer (50 mM Tris-HCl [pH 7.5], 100 mM KCl, 0.1 mM EDTA, 0.35% Nonidet P-40, 10 mM dithiothreitol [DTT], and protease inhibitors [Roche]) on ice for 30 min. For gel shift assays, 0.2 μl of cell extract was incubated in 10 μl binding buffer (10 mM Tris-HCl [pH 7.5], 100 mM KCl, 2 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride [PMSF], 15% glycerol, 5 mg of bovine serum albumin [BSA] per ml, protease inhibitors [Roche]) at room temperature for 20 min in the presence of 1 μg of sonicated salmon sperm DNA and 0.5 ng of a 32 P-labeled, double-stranded, high-affinity probe for BPV1 E2 binding site 9 (BS9; 5′-ACAAAGT ACCGTTGCCGGT CGAA-3′ [binding site is shown in bold]). ..

Article Title: Proteolytic cleavage and truncation of NDRG1 in human prostate cancer cells, but not normal prostate epithelial cells
Article Snippet: Cells were then lysed using RIPA lysis buffer [65 mM Tris/HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1% (v/v) Triton X-100, 0.1% (w/v) SDS, 0.5% (w/v) sodium deoxycholate, 10% (v/v) glycerol, protease inhibitor cocktail [Complete™ EDTA-free; Roche Applied Science] and PhosStop and sonicated on ice. .. Cell lysates were incubated on a rotator for 3 h at room temperature with 1.5 mg of Dynabeads protein G (Invitrogen) that had been pre-incubated with anti-NDRG1 (1:12.5, C-terminal) antibody diluted in PBS with 0.02% (v/v) Tween-20 for 3 h at room temperature.

Article Title: A role for caveolin-1 in desmoglein binding and desmosome dynamics
Article Snippet: .. For Tx-soluble proteins, cells were incubated on ice for 20 min in a 1% Tx-containing buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 5 mM EDTA), complete with protease inhibitors (Roche Diagnostics, Indianapolis, IN, USA) and phosphatase inhibitors (Sigma). ..

Article Title: Neutrophil Elastase-mediated proteolysis activates the anti-inflammatory cytokine IL-36 Receptor antagonist
Article Snippet: .. Cleavage assays, SDS PAGE and western blotting 1–2 μg of SUMO tagged IL-36Ra were incubated with supernatants from primary cells at 37 °C for 1 hour with or without protease inhibitors (IAA 50 μM), EDTA (500 μM), PMSF (1 mM), 1x PI Roche, α1 anti-trypsin (1 mg/ml) were used at concentrations outlined by the manufacturers. .. Incubations performed with recombinant proteases (proteinase 3, human neutrophil elastase, cathepsin G (Calbiochem)) were set up as recommended by manufacturers and incubated at 37 °C for up to 5 hours.

Article Title: DNA Methylation Analysis in the Intestinal Epithelium--Effect of Cell Separation on Gene Expression and Methylation Profile
Article Snippet: Alternative approaches use various digestive enzymes to disrupt the basement membrane and extracellular matrix of the lamina propria, sometimes after an initial EDTA/DTT incubation. .. Four pooled biopsy samples were then subjected to either chemical digestion using a combination of EDTA and DTT, or enzymatic digestion with Liberase (a blend of collagenase I/II and a neutral protease; Roche Applied Science) and hyaluronidase.

Mass Spectrometry:

Article Title: Proteolytic cleavage and truncation of NDRG1 in human prostate cancer cells, but not normal prostate epithelial cells
Article Snippet: Immunoprecipitation To obtain sufficient levels of protein to allow for analysis by MS, NDRG1 was immunoprecipitated from cells overexpressing NDRG1 . .. Cells were then lysed using RIPA lysis buffer [65 mM Tris/HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1% (v/v) Triton X-100, 0.1% (w/v) SDS, 0.5% (w/v) sodium deoxycholate, 10% (v/v) glycerol, protease inhibitor cocktail [Complete™ EDTA-free; Roche Applied Science] and PhosStop and sonicated on ice.

Article Title: Centriolar Satellites Control GABARAP Ubiquitination and GABARAP-Mediated Autophagy
Article Snippet: .. Ubiquitination mass spectrometry Cells were lysed in ice-cold buffer (Tris-HCl 20 mM pH6.8, 0.5% (w/v) Triton X-100, 150 mM NaCl, 5 mM EDTA, phosphatase inhibitor cocktail and mammalian protease inhibitor cocktail [Roche], and 20 mM N-Ethylmaleimide). .. The lysates were then clarified by centrifugation (5 min, full speed, 4°C) and the supernatants containing GFP-GABARAP ubiquitinated conjugates were subjected to immunoprecipitation at 4°C with GFP-TRAP beads for 2 hr.

Article Title: Neutrophil Elastase-mediated proteolysis activates the anti-inflammatory cytokine IL-36 Receptor antagonist
Article Snippet: Cleavage assays, SDS PAGE and western blotting 1–2 μg of SUMO tagged IL-36Ra were incubated with supernatants from primary cells at 37 °C for 1 hour with or without protease inhibitors (IAA 50 μM), EDTA (500 μM), PMSF (1 mM), 1x PI Roche, α1 anti-trypsin (1 mg/ml) were used at concentrations outlined by the manufacturers. .. Cleaved products were further analysed by in-house Liquid Chromatography Mass Spectrometry or analysed via N-terminal sequencing.

Modification:

Article Title: Interferon-? Reduces Cell Surface Expression of Annexin 2 and Suppresses the Invasive Capacity of Prostate Cancer Cells *Interferon-? Reduces Cell Surface Expression of Annexin 2 and Suppresses the Invasive Capacity of Prostate Cancer Cells * S⃞
Article Snippet: For avidin affinity chromatography, sulfo-NHS-SS-biotin-labeled cells were lysed in 1% octyl-β- d -glucopyranoside (OGP) in PBS, containing a protease inhibitor mixture with or without EDTA (Complete, Roche Applied Science). .. For avidin affinity chromatography, sulfo-NHS-SS-biotin-labeled cells were lysed in 1% octyl-β- d -glucopyranoside (OGP) in PBS, containing a protease inhibitor mixture with or without EDTA (Complete, Roche Applied Science).

Western Blot:

Article Title: Electroporation-mediated delivery of catalytic oligodeoxynucleotides for manipulation of vascular gene expression
Article Snippet: Paragraph title: Western blot analyses ... Cultured cells (100-mm dishes) were washed twice with PBS and lysed with 1 ml of 50 mM Tris (pH 7.5) containing 1% Triton X-100, 150 mM NaCl, 5 mM EDTA, and complete protease inhibitor (Roche).

Article Title: Neutrophil Elastase-mediated proteolysis activates the anti-inflammatory cytokine IL-36 Receptor antagonist
Article Snippet: .. Cleavage assays, SDS PAGE and western blotting 1–2 μg of SUMO tagged IL-36Ra were incubated with supernatants from primary cells at 37 °C for 1 hour with or without protease inhibitors (IAA 50 μM), EDTA (500 μM), PMSF (1 mM), 1x PI Roche, α1 anti-trypsin (1 mg/ml) were used at concentrations outlined by the manufacturers. .. Incubations performed with recombinant proteases (proteinase 3, human neutrophil elastase, cathepsin G (Calbiochem)) were set up as recommended by manufacturers and incubated at 37 °C for up to 5 hours.

Crystallization Assay:

Article Title: Crystal Structure of Penicillin-Binding Protein 3 (PBP3) from Escherichia coli
Article Snippet: Paragraph title: PBP388–165 : Production, Purification, Crystallization, Data Collection and Structure Refinement ... The strep-tag- PBP388–165 was isolated from E. coli C41(DE3) harbouring pDML2042 grown at 37°C in 2XYT medium in the presence of 0.5 mM IPTG for 3 h. The harvested cells were suspended in 40 ml of 100 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA containing a protease inhibitor cocktail (Roche) (buffer C), broken 5 times into a high-pressure homogenizer (Emulsiflex-C3 Avestin Inc.) and centrifuged at 25000 g for 40 min.

Lowry Assay:

Article Title: Interferon-? Reduces Cell Surface Expression of Annexin 2 and Suppresses the Invasive Capacity of Prostate Cancer Cells *Interferon-? Reduces Cell Surface Expression of Annexin 2 and Suppresses the Invasive Capacity of Prostate Cancer Cells * S⃞
Article Snippet: For avidin affinity chromatography, sulfo-NHS-SS-biotin-labeled cells were lysed in 1% octyl-β- d -glucopyranoside (OGP) in PBS, containing a protease inhibitor mixture with or without EDTA (Complete, Roche Applied Science). .. For avidin affinity chromatography, sulfo-NHS-SS-biotin-labeled cells were lysed in 1% octyl-β- d -glucopyranoside (OGP) in PBS, containing a protease inhibitor mixture with or without EDTA (Complete, Roche Applied Science).

Flow Cytometry:

Article Title: DNA Methylation Analysis in the Intestinal Epithelium--Effect of Cell Separation on Gene Expression and Methylation Profile
Article Snippet: Subsequent purification of IEC from accompanying mononuclear and stromal cells may be accomplished by density centrifugation or cell sorting using immunomagnetic beads or flow cytometry. .. Four pooled biopsy samples were then subjected to either chemical digestion using a combination of EDTA and DTT, or enzymatic digestion with Liberase (a blend of collagenase I/II and a neutral protease; Roche Applied Science) and hyaluronidase.

Immunoprecipitation:

Article Title: Proteolytic cleavage and truncation of NDRG1 in human prostate cancer cells, but not normal prostate epithelial cells
Article Snippet: Paragraph title: Immunoprecipitation ... Cells were then lysed using RIPA lysis buffer [65 mM Tris/HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1% (v/v) Triton X-100, 0.1% (w/v) SDS, 0.5% (w/v) sodium deoxycholate, 10% (v/v) glycerol, protease inhibitor cocktail [Complete™ EDTA-free; Roche Applied Science] and PhosStop and sonicated on ice.

Article Title: Centriolar Satellites Control GABARAP Ubiquitination and GABARAP-Mediated Autophagy
Article Snippet: Ubiquitination mass spectrometry Cells were lysed in ice-cold buffer (Tris-HCl 20 mM pH6.8, 0.5% (w/v) Triton X-100, 150 mM NaCl, 5 mM EDTA, phosphatase inhibitor cocktail and mammalian protease inhibitor cocktail [Roche], and 20 mM N-Ethylmaleimide). .. The lysates were then clarified by centrifugation (5 min, full speed, 4°C) and the supernatants containing GFP-GABARAP ubiquitinated conjugates were subjected to immunoprecipitation at 4°C with GFP-TRAP beads for 2 hr.

Protease Inhibitor:

Article Title: Proteolytic cleavage and truncation of NDRG1 in human prostate cancer cells, but not normal prostate epithelial cells
Article Snippet: .. Cells were then lysed using RIPA lysis buffer [65 mM Tris/HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1% (v/v) Triton X-100, 0.1% (w/v) SDS, 0.5% (w/v) sodium deoxycholate, 10% (v/v) glycerol, protease inhibitor cocktail [Complete™ EDTA-free; Roche Applied Science] and PhosStop and sonicated on ice. .. Cell lysates were incubated on a rotator for 3 h at room temperature with 1.5 mg of Dynabeads protein G (Invitrogen) that had been pre-incubated with anti-NDRG1 (1:12.5, C-terminal) antibody diluted in PBS with 0.02% (v/v) Tween-20 for 3 h at room temperature.

Article Title: Centriolar Satellites Control GABARAP Ubiquitination and GABARAP-Mediated Autophagy
Article Snippet: .. Ubiquitination mass spectrometry Cells were lysed in ice-cold buffer (Tris-HCl 20 mM pH6.8, 0.5% (w/v) Triton X-100, 150 mM NaCl, 5 mM EDTA, phosphatase inhibitor cocktail and mammalian protease inhibitor cocktail [Roche], and 20 mM N-Ethylmaleimide). .. The lysates were then clarified by centrifugation (5 min, full speed, 4°C) and the supernatants containing GFP-GABARAP ubiquitinated conjugates were subjected to immunoprecipitation at 4°C with GFP-TRAP beads for 2 hr.

Article Title: Electroporation-mediated delivery of catalytic oligodeoxynucleotides for manipulation of vascular gene expression
Article Snippet: .. Cultured cells (100-mm dishes) were washed twice with PBS and lysed with 1 ml of 50 mM Tris (pH 7.5) containing 1% Triton X-100, 150 mM NaCl, 5 mM EDTA, and complete protease inhibitor (Roche). ..

Article Title: Interferon-? Reduces Cell Surface Expression of Annexin 2 and Suppresses the Invasive Capacity of Prostate Cancer Cells *Interferon-? Reduces Cell Surface Expression of Annexin 2 and Suppresses the Invasive Capacity of Prostate Cancer Cells * S⃞
Article Snippet: .. For avidin affinity chromatography, sulfo-NHS-SS-biotin-labeled cells were lysed in 1% octyl-β- d -glucopyranoside (OGP) in PBS, containing a protease inhibitor mixture with or without EDTA (Complete, Roche Applied Science). ..

Article Title: The Structure and Regulation of Human Muscle α-Actinin
Article Snippet: .. After induction, cells were harvested by centrifugation and the cell pellet from one liter of high-density shaking cultures (OD600 ∼5 - 8 A.U.) were re-suspended in 50 ml of lysis buffer 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5mM EDTA, 0.1% Triton X-100 supplemented with antiproteases (Complete™ Protease Inhibitor Cocktail Tablets, Roche). ..

Article Title: Activation of MAPK/ERK signaling by Burkholderia pseudomallei cycle inhibiting factor (Cif)
Article Snippet: .. The composition of the lysis buffer was as follows: 25 mM Tris-HCl (pH 7.5), 100 mM NaCl, 2.5 mM EDTA, 2.5 mM EGTA, 20 mM NaF, 1 mM Na3 VO4 , 20 mM Sodium β-Glycerophosphate, 10 mM Sodium Pyrophosphate, 0.5% Triton X-100, Roche protease inhibitor cocktail and 0.1% β-Mercaptoethanol. ..

Article Title: Crystal Structure of Penicillin-Binding Protein 3 (PBP3) from Escherichia coli
Article Snippet: .. The strep-tag- PBP388–165 was isolated from E. coli C41(DE3) harbouring pDML2042 grown at 37°C in 2XYT medium in the presence of 0.5 mM IPTG for 3 h. The harvested cells were suspended in 40 ml of 100 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA containing a protease inhibitor cocktail (Roche) (buffer C), broken 5 times into a high-pressure homogenizer (Emulsiflex-C3 Avestin Inc.) and centrifuged at 25000 g for 40 min. ..

Cell Culture:

Article Title: A role for caveolin-1 in desmoglein binding and desmosome dynamics
Article Snippet: Paragraph title: Cell culture, drug treatment and protein extraction ... For Tx-soluble proteins, cells were incubated on ice for 20 min in a 1% Tx-containing buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 5 mM EDTA), complete with protease inhibitors (Roche Diagnostics, Indianapolis, IN, USA) and phosphatase inhibitors (Sigma).

Article Title: Electroporation-mediated delivery of catalytic oligodeoxynucleotides for manipulation of vascular gene expression
Article Snippet: .. Cultured cells (100-mm dishes) were washed twice with PBS and lysed with 1 ml of 50 mM Tris (pH 7.5) containing 1% Triton X-100, 150 mM NaCl, 5 mM EDTA, and complete protease inhibitor (Roche). ..

SDS Page:

Article Title: Centriolar Satellites Control GABARAP Ubiquitination and GABARAP-Mediated Autophagy
Article Snippet: Ubiquitination mass spectrometry Cells were lysed in ice-cold buffer (Tris-HCl 20 mM pH6.8, 0.5% (w/v) Triton X-100, 150 mM NaCl, 5 mM EDTA, phosphatase inhibitor cocktail and mammalian protease inhibitor cocktail [Roche], and 20 mM N-Ethylmaleimide). .. Immunoprecipitates were washed three times with stringent denaturing washing buffer (8 M Urea, 1% SDS in PBS) at room temperature to remove GABARAP binding partners and then once with 10 mM Tris pH 7.5 before preparation for SDS-PAGE.

Article Title: Neutrophil Elastase-mediated proteolysis activates the anti-inflammatory cytokine IL-36 Receptor antagonist
Article Snippet: .. Cleavage assays, SDS PAGE and western blotting 1–2 μg of SUMO tagged IL-36Ra were incubated with supernatants from primary cells at 37 °C for 1 hour with or without protease inhibitors (IAA 50 μM), EDTA (500 μM), PMSF (1 mM), 1x PI Roche, α1 anti-trypsin (1 mg/ml) were used at concentrations outlined by the manufacturers. .. Incubations performed with recombinant proteases (proteinase 3, human neutrophil elastase, cathepsin G (Calbiochem)) were set up as recommended by manufacturers and incubated at 37 °C for up to 5 hours.

Polymerase Chain Reaction:

Article Title: Maintaining mRNA Integrity during Decalcification of Mineralized Tissues
Article Snippet: Quantitative PCR RNA isolated from EDTA or RNAlater /EDTA decalcified whole tibiae (500 ng total RNA) was reverse-transcribed as per the manufacturer’s protocol in a 20µL reaction using the Transcriptor High Fidelity cDNA Synthesis Kit (Roche, Germany). .. Quantitative PCR (qPCR) reactions containing 2.5 ng cDNA were prepared to 5µL in 384 well PCR plates (Roche) using the LightCycler® 480 SYBR Green I Master (Roche) as per the manufacturer’s instructions and run on a LC480 II thermo cycler (Roche).

Article Title: Crystal Structure of Penicillin-Binding Protein 3 (PBP3) from Escherichia coli
Article Snippet: The PCR fragment was cloned into the pJet1.2/blunt cloning vector (Fermentas), sequenced, digested with XmaI and BamHI and inserted into the same sites of plasmid pET-52b(+). .. The strep-tag- PBP388–165 was isolated from E. coli C41(DE3) harbouring pDML2042 grown at 37°C in 2XYT medium in the presence of 0.5 mM IPTG for 3 h. The harvested cells were suspended in 40 ml of 100 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA containing a protease inhibitor cocktail (Roche) (buffer C), broken 5 times into a high-pressure homogenizer (Emulsiflex-C3 Avestin Inc.) and centrifuged at 25000 g for 40 min.

Sonication:

Article Title: Effective Formation of the Segregation-Competent Complex Determines Successful Partitioning of the Bovine Papillomavirus Genome during Cell Division ▿Effective Formation of the Segregation-Competent Complex Determines Successful Partitioning of the Bovine Papillomavirus Genome during Cell Division ▿ †
Article Snippet: .. Briefly, COS-7 cell extracts were transfected with 1 μg expression plasmid pCG and lysed in 100 μl of lysis buffer (50 mM Tris-HCl [pH 7.5], 100 mM KCl, 0.1 mM EDTA, 0.35% Nonidet P-40, 10 mM dithiothreitol [DTT], and protease inhibitors [Roche]) on ice for 30 min. For gel shift assays, 0.2 μl of cell extract was incubated in 10 μl binding buffer (10 mM Tris-HCl [pH 7.5], 100 mM KCl, 2 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride [PMSF], 15% glycerol, 5 mg of bovine serum albumin [BSA] per ml, protease inhibitors [Roche]) at room temperature for 20 min in the presence of 1 μg of sonicated salmon sperm DNA and 0.5 ng of a 32 P-labeled, double-stranded, high-affinity probe for BPV1 E2 binding site 9 (BS9; 5′-ACAAAGT ACCGTTGCCGGT CGAA-3′ [binding site is shown in bold]). ..

Article Title: Proteolytic cleavage and truncation of NDRG1 in human prostate cancer cells, but not normal prostate epithelial cells
Article Snippet: .. Cells were then lysed using RIPA lysis buffer [65 mM Tris/HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1% (v/v) Triton X-100, 0.1% (w/v) SDS, 0.5% (w/v) sodium deoxycholate, 10% (v/v) glycerol, protease inhibitor cocktail [Complete™ EDTA-free; Roche Applied Science] and PhosStop and sonicated on ice. .. Cell lysates were incubated on a rotator for 3 h at room temperature with 1.5 mg of Dynabeads protein G (Invitrogen) that had been pre-incubated with anti-NDRG1 (1:12.5, C-terminal) antibody diluted in PBS with 0.02% (v/v) Tween-20 for 3 h at room temperature.

Recombinant:

Article Title: The Structure and Regulation of Human Muscle α-Actinin
Article Snippet: Protein Expression and Purification for Biochemical Studies Full-Length α-Actinin-2 Constructs. α-actinin-2 constructs (wild-type, R268E/I269E/L273E, R163E, R163E/R169E, and R163E/R169E/R192E) were expressed using a recombinant plasmid pET3d containing the full-length gene with a His6 -Tag. .. After induction, cells were harvested by centrifugation and the cell pellet from one liter of high-density shaking cultures (OD600 ∼5 - 8 A.U.) were re-suspended in 50 ml of lysis buffer 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5mM EDTA, 0.1% Triton X-100 supplemented with antiproteases (Complete™ Protease Inhibitor Cocktail Tablets, Roche).

Article Title: Neutrophil Elastase-mediated proteolysis activates the anti-inflammatory cytokine IL-36 Receptor antagonist
Article Snippet: Cleavage assays, SDS PAGE and western blotting 1–2 μg of SUMO tagged IL-36Ra were incubated with supernatants from primary cells at 37 °C for 1 hour with or without protease inhibitors (IAA 50 μM), EDTA (500 μM), PMSF (1 mM), 1x PI Roche, α1 anti-trypsin (1 mg/ml) were used at concentrations outlined by the manufacturers. .. Incubations performed with recombinant proteases (proteinase 3, human neutrophil elastase, cathepsin G (Calbiochem)) were set up as recommended by manufacturers and incubated at 37 °C for up to 5 hours.

DC Protein Assay:

Article Title: Interferon-? Reduces Cell Surface Expression of Annexin 2 and Suppresses the Invasive Capacity of Prostate Cancer Cells *Interferon-? Reduces Cell Surface Expression of Annexin 2 and Suppresses the Invasive Capacity of Prostate Cancer Cells * S⃞
Article Snippet: For avidin affinity chromatography, sulfo-NHS-SS-biotin-labeled cells were lysed in 1% octyl-β- d -glucopyranoside (OGP) in PBS, containing a protease inhibitor mixture with or without EDTA (Complete, Roche Applied Science). .. For avidin affinity chromatography, sulfo-NHS-SS-biotin-labeled cells were lysed in 1% octyl-β- d -glucopyranoside (OGP) in PBS, containing a protease inhibitor mixture with or without EDTA (Complete, Roche Applied Science).

In Vivo:

Article Title: Electroporation-mediated delivery of catalytic oligodeoxynucleotides for manipulation of vascular gene expression
Article Snippet: Cultured cells (100-mm dishes) were washed twice with PBS and lysed with 1 ml of 50 mM Tris (pH 7.5) containing 1% Triton X-100, 150 mM NaCl, 5 mM EDTA, and complete protease inhibitor (Roche). .. For in vivo analyses, mesenteric neurovascular bundles were excised from rats and frozen in liquid nitrogen before mechanical disruption using a drill press as previously described ( ).

Isolation:

Article Title: Maintaining mRNA Integrity during Decalcification of Mineralized Tissues
Article Snippet: .. Quantitative PCR RNA isolated from EDTA or RNAlater /EDTA decalcified whole tibiae (500 ng total RNA) was reverse-transcribed as per the manufacturer’s protocol in a 20µL reaction using the Transcriptor High Fidelity cDNA Synthesis Kit (Roche, Germany). .. Quantitative PCR (qPCR) reactions containing 2.5 ng cDNA were prepared to 5µL in 384 well PCR plates (Roche) using the LightCycler® 480 SYBR Green I Master (Roche) as per the manufacturer’s instructions and run on a LC480 II thermo cycler (Roche).

Article Title: Crystal Structure of Penicillin-Binding Protein 3 (PBP3) from Escherichia coli
Article Snippet: .. The strep-tag- PBP388–165 was isolated from E. coli C41(DE3) harbouring pDML2042 grown at 37°C in 2XYT medium in the presence of 0.5 mM IPTG for 3 h. The harvested cells were suspended in 40 ml of 100 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA containing a protease inhibitor cocktail (Roche) (buffer C), broken 5 times into a high-pressure homogenizer (Emulsiflex-C3 Avestin Inc.) and centrifuged at 25000 g for 40 min. ..

Article Title: DNA Methylation Analysis in the Intestinal Epithelium--Effect of Cell Separation on Gene Expression and Methylation Profile
Article Snippet: Paragraph title: Effectiveness of Isolation Procedures ... Four pooled biopsy samples were then subjected to either chemical digestion using a combination of EDTA and DTT, or enzymatic digestion with Liberase (a blend of collagenase I/II and a neutral protease; Roche Applied Science) and hyaluronidase.

Transfection:

Article Title: Effective Formation of the Segregation-Competent Complex Determines Successful Partitioning of the Bovine Papillomavirus Genome during Cell Division ▿Effective Formation of the Segregation-Competent Complex Determines Successful Partitioning of the Bovine Papillomavirus Genome during Cell Division ▿ †
Article Snippet: .. Briefly, COS-7 cell extracts were transfected with 1 μg expression plasmid pCG and lysed in 100 μl of lysis buffer (50 mM Tris-HCl [pH 7.5], 100 mM KCl, 0.1 mM EDTA, 0.35% Nonidet P-40, 10 mM dithiothreitol [DTT], and protease inhibitors [Roche]) on ice for 30 min. For gel shift assays, 0.2 μl of cell extract was incubated in 10 μl binding buffer (10 mM Tris-HCl [pH 7.5], 100 mM KCl, 2 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride [PMSF], 15% glycerol, 5 mg of bovine serum albumin [BSA] per ml, protease inhibitors [Roche]) at room temperature for 20 min in the presence of 1 μg of sonicated salmon sperm DNA and 0.5 ng of a 32 P-labeled, double-stranded, high-affinity probe for BPV1 E2 binding site 9 (BS9; 5′-ACAAAGT ACCGTTGCCGGT CGAA-3′ [binding site is shown in bold]). ..

Article Title: Proteolytic cleavage and truncation of NDRG1 in human prostate cancer cells, but not normal prostate epithelial cells
Article Snippet: Namely, human prostate cancer cells (PC3MM) transfected with tetracycline-inducible (TET-ON) human NDRG1 (pcDNA5/TO/Flag-Drg-1) were used. .. Cells were then lysed using RIPA lysis buffer [65 mM Tris/HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1% (v/v) Triton X-100, 0.1% (w/v) SDS, 0.5% (w/v) sodium deoxycholate, 10% (v/v) glycerol, protease inhibitor cocktail [Complete™ EDTA-free; Roche Applied Science] and PhosStop and sonicated on ice.

Article Title: The Structure and Regulation of Human Muscle α-Actinin
Article Snippet: The plasmids were transfected into suitable E. coli host strains for high-level expression that were defined based on protein expression optimization screened for each constructs. .. After induction, cells were harvested by centrifugation and the cell pellet from one liter of high-density shaking cultures (OD600 ∼5 - 8 A.U.) were re-suspended in 50 ml of lysis buffer 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5mM EDTA, 0.1% Triton X-100 supplemented with antiproteases (Complete™ Protease Inhibitor Cocktail Tablets, Roche).

Electrophoretic Mobility Shift Assay:

Article Title: Effective Formation of the Segregation-Competent Complex Determines Successful Partitioning of the Bovine Papillomavirus Genome during Cell Division ▿Effective Formation of the Segregation-Competent Complex Determines Successful Partitioning of the Bovine Papillomavirus Genome during Cell Division ▿ †
Article Snippet: .. Briefly, COS-7 cell extracts were transfected with 1 μg expression plasmid pCG and lysed in 100 μl of lysis buffer (50 mM Tris-HCl [pH 7.5], 100 mM KCl, 0.1 mM EDTA, 0.35% Nonidet P-40, 10 mM dithiothreitol [DTT], and protease inhibitors [Roche]) on ice for 30 min. For gel shift assays, 0.2 μl of cell extract was incubated in 10 μl binding buffer (10 mM Tris-HCl [pH 7.5], 100 mM KCl, 2 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride [PMSF], 15% glycerol, 5 mg of bovine serum albumin [BSA] per ml, protease inhibitors [Roche]) at room temperature for 20 min in the presence of 1 μg of sonicated salmon sperm DNA and 0.5 ng of a 32 P-labeled, double-stranded, high-affinity probe for BPV1 E2 binding site 9 (BS9; 5′-ACAAAGT ACCGTTGCCGGT CGAA-3′ [binding site is shown in bold]). ..

Avidin-Biotin Assay:

Article Title: Interferon-? Reduces Cell Surface Expression of Annexin 2 and Suppresses the Invasive Capacity of Prostate Cancer Cells *Interferon-? Reduces Cell Surface Expression of Annexin 2 and Suppresses the Invasive Capacity of Prostate Cancer Cells * S⃞
Article Snippet: .. For avidin affinity chromatography, sulfo-NHS-SS-biotin-labeled cells were lysed in 1% octyl-β- d -glucopyranoside (OGP) in PBS, containing a protease inhibitor mixture with or without EDTA (Complete, Roche Applied Science). ..

Sequencing:

Article Title: Neutrophil Elastase-mediated proteolysis activates the anti-inflammatory cytokine IL-36 Receptor antagonist
Article Snippet: Cleavage assays, SDS PAGE and western blotting 1–2 μg of SUMO tagged IL-36Ra were incubated with supernatants from primary cells at 37 °C for 1 hour with or without protease inhibitors (IAA 50 μM), EDTA (500 μM), PMSF (1 mM), 1x PI Roche, α1 anti-trypsin (1 mg/ml) were used at concentrations outlined by the manufacturers. .. Cleaved products were further analysed by in-house Liquid Chromatography Mass Spectrometry or analysed via N-terminal sequencing.

Protein Extraction:

Article Title: A role for caveolin-1 in desmoglein binding and desmosome dynamics
Article Snippet: Paragraph title: Cell culture, drug treatment and protein extraction ... For Tx-soluble proteins, cells were incubated on ice for 20 min in a 1% Tx-containing buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 5 mM EDTA), complete with protease inhibitors (Roche Diagnostics, Indianapolis, IN, USA) and phosphatase inhibitors (Sigma).

Positron Emission Tomography:

Article Title: Crystal Structure of Penicillin-Binding Protein 3 (PBP3) from Escherichia coli
Article Snippet: The PCR fragment was cloned into the pJet1.2/blunt cloning vector (Fermentas), sequenced, digested with XmaI and BamHI and inserted into the same sites of plasmid pET-52b(+). .. The strep-tag- PBP388–165 was isolated from E. coli C41(DE3) harbouring pDML2042 grown at 37°C in 2XYT medium in the presence of 0.5 mM IPTG for 3 h. The harvested cells were suspended in 40 ml of 100 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA containing a protease inhibitor cocktail (Roche) (buffer C), broken 5 times into a high-pressure homogenizer (Emulsiflex-C3 Avestin Inc.) and centrifuged at 25000 g for 40 min.

Labeling:

Article Title: Interferon-? Reduces Cell Surface Expression of Annexin 2 and Suppresses the Invasive Capacity of Prostate Cancer Cells *Interferon-? Reduces Cell Surface Expression of Annexin 2 and Suppresses the Invasive Capacity of Prostate Cancer Cells * S⃞
Article Snippet: Surface Labeling, Enrichment, and Separation of Membrane Proteins —Vectorial labeling with sulfonated succimidyl ester derivatives of biotin ( ) was performed prior to interferon exposure or at the end of each treatment period. .. For avidin affinity chromatography, sulfo-NHS-SS-biotin-labeled cells were lysed in 1% octyl-β- d -glucopyranoside (OGP) in PBS, containing a protease inhibitor mixture with or without EDTA (Complete, Roche Applied Science).

Polyacrylamide Gel Electrophoresis:

Article Title: Effective Formation of the Segregation-Competent Complex Determines Successful Partitioning of the Bovine Papillomavirus Genome during Cell Division ▿Effective Formation of the Segregation-Competent Complex Determines Successful Partitioning of the Bovine Papillomavirus Genome during Cell Division ▿ †
Article Snippet: Briefly, COS-7 cell extracts were transfected with 1 μg expression plasmid pCG and lysed in 100 μl of lysis buffer (50 mM Tris-HCl [pH 7.5], 100 mM KCl, 0.1 mM EDTA, 0.35% Nonidet P-40, 10 mM dithiothreitol [DTT], and protease inhibitors [Roche]) on ice for 30 min. For gel shift assays, 0.2 μl of cell extract was incubated in 10 μl binding buffer (10 mM Tris-HCl [pH 7.5], 100 mM KCl, 2 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride [PMSF], 15% glycerol, 5 mg of bovine serum albumin [BSA] per ml, protease inhibitors [Roche]) at room temperature for 20 min in the presence of 1 μg of sonicated salmon sperm DNA and 0.5 ng of a 32 P-labeled, double-stranded, high-affinity probe for BPV1 E2 binding site 9 (BS9; 5′-ACAAAGT ACCGTTGCCGGT CGAA-3′ [binding site is shown in bold]). .. Protein-DNA complexes were resolved by 6% PAGE (acrylamide- N , N -methylene-bisacrylamide; 80:1) in 0.25× Tris-borate-EDTA buffer (TBE).

Article Title: Crystal Structure of Penicillin-Binding Protein 3 (PBP3) from Escherichia coli
Article Snippet: The strep-tag- PBP388–165 was isolated from E. coli C41(DE3) harbouring pDML2042 grown at 37°C in 2XYT medium in the presence of 0.5 mM IPTG for 3 h. The harvested cells were suspended in 40 ml of 100 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA containing a protease inhibitor cocktail (Roche) (buffer C), broken 5 times into a high-pressure homogenizer (Emulsiflex-C3 Avestin Inc.) and centrifuged at 25000 g for 40 min. .. The fractions of interest (5 ml) were dialyzed against 2 L of buffer C with a 3,500 Dalton cut off membrane and analyzed by SDS-18% PAGE.

Staining:

Article Title: Centriolar Satellites Control GABARAP Ubiquitination and GABARAP-Mediated Autophagy
Article Snippet: Ubiquitination mass spectrometry Cells were lysed in ice-cold buffer (Tris-HCl 20 mM pH6.8, 0.5% (w/v) Triton X-100, 150 mM NaCl, 5 mM EDTA, phosphatase inhibitor cocktail and mammalian protease inhibitor cocktail [Roche], and 20 mM N-Ethylmaleimide). .. Proteins were resolved by SDS-PAGE, fixed, and stained with GelCode, and gel slices subjected to tryptic digestion and mass spectrometry analysis (below).

Purification:

Article Title: Interferon-? Reduces Cell Surface Expression of Annexin 2 and Suppresses the Invasive Capacity of Prostate Cancer Cells *Interferon-? Reduces Cell Surface Expression of Annexin 2 and Suppresses the Invasive Capacity of Prostate Cancer Cells * S⃞
Article Snippet: For avidin affinity chromatography, sulfo-NHS-SS-biotin-labeled cells were lysed in 1% octyl-β- d -glucopyranoside (OGP) in PBS, containing a protease inhibitor mixture with or without EDTA (Complete, Roche Applied Science). .. The OGP-solubilized SS-biotin-labeled membrane fractions were purified by avidin affinity chromatography as described previously ( ).

Article Title: The Structure and Regulation of Human Muscle α-Actinin
Article Snippet: Paragraph title: Protein Expression and Purification for Biochemical Studies ... After induction, cells were harvested by centrifugation and the cell pellet from one liter of high-density shaking cultures (OD600 ∼5 - 8 A.U.) were re-suspended in 50 ml of lysis buffer 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5mM EDTA, 0.1% Triton X-100 supplemented with antiproteases (Complete™ Protease Inhibitor Cocktail Tablets, Roche).

Article Title: Crystal Structure of Penicillin-Binding Protein 3 (PBP3) from Escherichia coli
Article Snippet: Paragraph title: PBP388–165 : Production, Purification, Crystallization, Data Collection and Structure Refinement ... The strep-tag- PBP388–165 was isolated from E. coli C41(DE3) harbouring pDML2042 grown at 37°C in 2XYT medium in the presence of 0.5 mM IPTG for 3 h. The harvested cells were suspended in 40 ml of 100 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA containing a protease inhibitor cocktail (Roche) (buffer C), broken 5 times into a high-pressure homogenizer (Emulsiflex-C3 Avestin Inc.) and centrifuged at 25000 g for 40 min.

Article Title: DNA Methylation Analysis in the Intestinal Epithelium--Effect of Cell Separation on Gene Expression and Methylation Profile
Article Snippet: Subsequent purification of IEC from accompanying mononuclear and stromal cells may be accomplished by density centrifugation or cell sorting using immunomagnetic beads or flow cytometry. .. Four pooled biopsy samples were then subjected to either chemical digestion using a combination of EDTA and DTT, or enzymatic digestion with Liberase (a blend of collagenase I/II and a neutral protease; Roche Applied Science) and hyaluronidase.

Liquid Chromatography:

Article Title: Neutrophil Elastase-mediated proteolysis activates the anti-inflammatory cytokine IL-36 Receptor antagonist
Article Snippet: Cleavage assays, SDS PAGE and western blotting 1–2 μg of SUMO tagged IL-36Ra were incubated with supernatants from primary cells at 37 °C for 1 hour with or without protease inhibitors (IAA 50 μM), EDTA (500 μM), PMSF (1 mM), 1x PI Roche, α1 anti-trypsin (1 mg/ml) were used at concentrations outlined by the manufacturers. .. Cleaved products were further analysed by in-house Liquid Chromatography Mass Spectrometry or analysed via N-terminal sequencing.

Plasmid Preparation:

Article Title: Effective Formation of the Segregation-Competent Complex Determines Successful Partitioning of the Bovine Papillomavirus Genome during Cell Division ▿Effective Formation of the Segregation-Competent Complex Determines Successful Partitioning of the Bovine Papillomavirus Genome during Cell Division ▿ †
Article Snippet: .. Briefly, COS-7 cell extracts were transfected with 1 μg expression plasmid pCG and lysed in 100 μl of lysis buffer (50 mM Tris-HCl [pH 7.5], 100 mM KCl, 0.1 mM EDTA, 0.35% Nonidet P-40, 10 mM dithiothreitol [DTT], and protease inhibitors [Roche]) on ice for 30 min. For gel shift assays, 0.2 μl of cell extract was incubated in 10 μl binding buffer (10 mM Tris-HCl [pH 7.5], 100 mM KCl, 2 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride [PMSF], 15% glycerol, 5 mg of bovine serum albumin [BSA] per ml, protease inhibitors [Roche]) at room temperature for 20 min in the presence of 1 μg of sonicated salmon sperm DNA and 0.5 ng of a 32 P-labeled, double-stranded, high-affinity probe for BPV1 E2 binding site 9 (BS9; 5′-ACAAAGT ACCGTTGCCGGT CGAA-3′ [binding site is shown in bold]). ..

Article Title: The Structure and Regulation of Human Muscle α-Actinin
Article Snippet: Protein Expression and Purification for Biochemical Studies Full-Length α-Actinin-2 Constructs. α-actinin-2 constructs (wild-type, R268E/I269E/L273E, R163E, R163E/R169E, and R163E/R169E/R192E) were expressed using a recombinant plasmid pET3d containing the full-length gene with a His6 -Tag. .. After induction, cells were harvested by centrifugation and the cell pellet from one liter of high-density shaking cultures (OD600 ∼5 - 8 A.U.) were re-suspended in 50 ml of lysis buffer 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5mM EDTA, 0.1% Triton X-100 supplemented with antiproteases (Complete™ Protease Inhibitor Cocktail Tablets, Roche).

Article Title: Crystal Structure of Penicillin-Binding Protein 3 (PBP3) from Escherichia coli
Article Snippet: The resulting plasmid pDML2042 codes for the PBP388–165 with an N-terminal strep-tag. .. The strep-tag- PBP388–165 was isolated from E. coli C41(DE3) harbouring pDML2042 grown at 37°C in 2XYT medium in the presence of 0.5 mM IPTG for 3 h. The harvested cells were suspended in 40 ml of 100 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA containing a protease inhibitor cocktail (Roche) (buffer C), broken 5 times into a high-pressure homogenizer (Emulsiflex-C3 Avestin Inc.) and centrifuged at 25000 g for 40 min.

Real-time Polymerase Chain Reaction:

Article Title: Maintaining mRNA Integrity during Decalcification of Mineralized Tissues
Article Snippet: .. Quantitative PCR RNA isolated from EDTA or RNAlater /EDTA decalcified whole tibiae (500 ng total RNA) was reverse-transcribed as per the manufacturer’s protocol in a 20µL reaction using the Transcriptor High Fidelity cDNA Synthesis Kit (Roche, Germany). .. Quantitative PCR (qPCR) reactions containing 2.5 ng cDNA were prepared to 5µL in 384 well PCR plates (Roche) using the LightCycler® 480 SYBR Green I Master (Roche) as per the manufacturer’s instructions and run on a LC480 II thermo cycler (Roche).

Binding Assay:

Article Title: Effective Formation of the Segregation-Competent Complex Determines Successful Partitioning of the Bovine Papillomavirus Genome during Cell Division ▿Effective Formation of the Segregation-Competent Complex Determines Successful Partitioning of the Bovine Papillomavirus Genome during Cell Division ▿ †
Article Snippet: .. Briefly, COS-7 cell extracts were transfected with 1 μg expression plasmid pCG and lysed in 100 μl of lysis buffer (50 mM Tris-HCl [pH 7.5], 100 mM KCl, 0.1 mM EDTA, 0.35% Nonidet P-40, 10 mM dithiothreitol [DTT], and protease inhibitors [Roche]) on ice for 30 min. For gel shift assays, 0.2 μl of cell extract was incubated in 10 μl binding buffer (10 mM Tris-HCl [pH 7.5], 100 mM KCl, 2 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride [PMSF], 15% glycerol, 5 mg of bovine serum albumin [BSA] per ml, protease inhibitors [Roche]) at room temperature for 20 min in the presence of 1 μg of sonicated salmon sperm DNA and 0.5 ng of a 32 P-labeled, double-stranded, high-affinity probe for BPV1 E2 binding site 9 (BS9; 5′-ACAAAGT ACCGTTGCCGGT CGAA-3′ [binding site is shown in bold]). ..

Article Title: Centriolar Satellites Control GABARAP Ubiquitination and GABARAP-Mediated Autophagy
Article Snippet: Ubiquitination mass spectrometry Cells were lysed in ice-cold buffer (Tris-HCl 20 mM pH6.8, 0.5% (w/v) Triton X-100, 150 mM NaCl, 5 mM EDTA, phosphatase inhibitor cocktail and mammalian protease inhibitor cocktail [Roche], and 20 mM N-Ethylmaleimide). .. Immunoprecipitates were washed three times with stringent denaturing washing buffer (8 M Urea, 1% SDS in PBS) at room temperature to remove GABARAP binding partners and then once with 10 mM Tris pH 7.5 before preparation for SDS-PAGE.

Selection:

Article Title: DNA Methylation Analysis in the Intestinal Epithelium--Effect of Cell Separation on Gene Expression and Methylation Profile
Article Snippet: Four pooled biopsy samples were then subjected to either chemical digestion using a combination of EDTA and DTT, or enzymatic digestion with Liberase (a blend of collagenase I/II and a neutral protease; Roche Applied Science) and hyaluronidase. .. Resulting cell suspensions were then used for immunomagnetic bead based positive selection of IEC using antibodies against the epithelial cell specific adhesion molecule CD326.

Affinity Chromatography:

Article Title: Interferon-? Reduces Cell Surface Expression of Annexin 2 and Suppresses the Invasive Capacity of Prostate Cancer Cells *Interferon-? Reduces Cell Surface Expression of Annexin 2 and Suppresses the Invasive Capacity of Prostate Cancer Cells * S⃞
Article Snippet: .. For avidin affinity chromatography, sulfo-NHS-SS-biotin-labeled cells were lysed in 1% octyl-β- d -glucopyranoside (OGP) in PBS, containing a protease inhibitor mixture with or without EDTA (Complete, Roche Applied Science). ..

Mobility Shift:

Article Title: Effective Formation of the Segregation-Competent Complex Determines Successful Partitioning of the Bovine Papillomavirus Genome during Cell Division ▿Effective Formation of the Segregation-Competent Complex Determines Successful Partitioning of the Bovine Papillomavirus Genome during Cell Division ▿ †
Article Snippet: Paragraph title: Mobility shift assay. ... Briefly, COS-7 cell extracts were transfected with 1 μg expression plasmid pCG and lysed in 100 μl of lysis buffer (50 mM Tris-HCl [pH 7.5], 100 mM KCl, 0.1 mM EDTA, 0.35% Nonidet P-40, 10 mM dithiothreitol [DTT], and protease inhibitors [Roche]) on ice for 30 min. For gel shift assays, 0.2 μl of cell extract was incubated in 10 μl binding buffer (10 mM Tris-HCl [pH 7.5], 100 mM KCl, 2 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride [PMSF], 15% glycerol, 5 mg of bovine serum albumin [BSA] per ml, protease inhibitors [Roche]) at room temperature for 20 min in the presence of 1 μg of sonicated salmon sperm DNA and 0.5 ng of a 32 P-labeled, double-stranded, high-affinity probe for BPV1 E2 binding site 9 (BS9; 5′-ACAAAGT ACCGTTGCCGGT CGAA-3′ [binding site is shown in bold]).

Lysis:

Article Title: Effective Formation of the Segregation-Competent Complex Determines Successful Partitioning of the Bovine Papillomavirus Genome during Cell Division ▿Effective Formation of the Segregation-Competent Complex Determines Successful Partitioning of the Bovine Papillomavirus Genome during Cell Division ▿ †
Article Snippet: .. Briefly, COS-7 cell extracts were transfected with 1 μg expression plasmid pCG and lysed in 100 μl of lysis buffer (50 mM Tris-HCl [pH 7.5], 100 mM KCl, 0.1 mM EDTA, 0.35% Nonidet P-40, 10 mM dithiothreitol [DTT], and protease inhibitors [Roche]) on ice for 30 min. For gel shift assays, 0.2 μl of cell extract was incubated in 10 μl binding buffer (10 mM Tris-HCl [pH 7.5], 100 mM KCl, 2 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride [PMSF], 15% glycerol, 5 mg of bovine serum albumin [BSA] per ml, protease inhibitors [Roche]) at room temperature for 20 min in the presence of 1 μg of sonicated salmon sperm DNA and 0.5 ng of a 32 P-labeled, double-stranded, high-affinity probe for BPV1 E2 binding site 9 (BS9; 5′-ACAAAGT ACCGTTGCCGGT CGAA-3′ [binding site is shown in bold]). ..

Article Title: Proteolytic cleavage and truncation of NDRG1 in human prostate cancer cells, but not normal prostate epithelial cells
Article Snippet: .. Cells were then lysed using RIPA lysis buffer [65 mM Tris/HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1% (v/v) Triton X-100, 0.1% (w/v) SDS, 0.5% (w/v) sodium deoxycholate, 10% (v/v) glycerol, protease inhibitor cocktail [Complete™ EDTA-free; Roche Applied Science] and PhosStop and sonicated on ice. .. Cell lysates were incubated on a rotator for 3 h at room temperature with 1.5 mg of Dynabeads protein G (Invitrogen) that had been pre-incubated with anti-NDRG1 (1:12.5, C-terminal) antibody diluted in PBS with 0.02% (v/v) Tween-20 for 3 h at room temperature.

Article Title: Electroporation-mediated delivery of catalytic oligodeoxynucleotides for manipulation of vascular gene expression
Article Snippet: Cultured cells (100-mm dishes) were washed twice with PBS and lysed with 1 ml of 50 mM Tris (pH 7.5) containing 1% Triton X-100, 150 mM NaCl, 5 mM EDTA, and complete protease inhibitor (Roche). .. The ground tissue was suspended in Promega lysis buffer containing 1 mM dithiothreitol and subjected to three freeze-thaw cycles.

Article Title: The Structure and Regulation of Human Muscle α-Actinin
Article Snippet: .. After induction, cells were harvested by centrifugation and the cell pellet from one liter of high-density shaking cultures (OD600 ∼5 - 8 A.U.) were re-suspended in 50 ml of lysis buffer 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5mM EDTA, 0.1% Triton X-100 supplemented with antiproteases (Complete™ Protease Inhibitor Cocktail Tablets, Roche). ..

Article Title: Activation of MAPK/ERK signaling by Burkholderia pseudomallei cycle inhibiting factor (Cif)
Article Snippet: .. The composition of the lysis buffer was as follows: 25 mM Tris-HCl (pH 7.5), 100 mM NaCl, 2.5 mM EDTA, 2.5 mM EGTA, 20 mM NaF, 1 mM Na3 VO4 , 20 mM Sodium β-Glycerophosphate, 10 mM Sodium Pyrophosphate, 0.5% Triton X-100, Roche protease inhibitor cocktail and 0.1% β-Mercaptoethanol. ..

Strep-tag:

Article Title: Crystal Structure of Penicillin-Binding Protein 3 (PBP3) from Escherichia coli
Article Snippet: .. The strep-tag- PBP388–165 was isolated from E. coli C41(DE3) harbouring pDML2042 grown at 37°C in 2XYT medium in the presence of 0.5 mM IPTG for 3 h. The harvested cells were suspended in 40 ml of 100 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA containing a protease inhibitor cocktail (Roche) (buffer C), broken 5 times into a high-pressure homogenizer (Emulsiflex-C3 Avestin Inc.) and centrifuged at 25000 g for 40 min. ..

FACS:

Article Title: DNA Methylation Analysis in the Intestinal Epithelium--Effect of Cell Separation on Gene Expression and Methylation Profile
Article Snippet: Subsequent purification of IEC from accompanying mononuclear and stromal cells may be accomplished by density centrifugation or cell sorting using immunomagnetic beads or flow cytometry. .. Four pooled biopsy samples were then subjected to either chemical digestion using a combination of EDTA and DTT, or enzymatic digestion with Liberase (a blend of collagenase I/II and a neutral protease; Roche Applied Science) and hyaluronidase.

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  • edta  (Roche)
    99
    Roche edta
    Cytokeratin 8 and CD3γ copy numbers in intestinal epithelial cells. A) FACS analysis of T cell contamination. Positive selection of IEC was performed using CD326 Microbeads followed by staining using T-cell receptor antibodies and Pacific Blue Annexin V to detect apoptotic cells. Panel A1 shows an example of IEC isolated using the enzyme based protocol, panel A2 using the <t>EDTA/DTT</t> based protocol. T-cell receptor positive cells are located in quadrant Q1 and Q2. Analysis of absolute mRNA copy numbers of cytokeratin 8 and CD3γ in intestinal epithelial cells separated by positive selection using CD326 Microbeads cells. B) Expression of mRNA copy numbers of Cytokeratin 8 and CD3γ. Data is expressed as box and whisker plots. The length of the boxes represents the interquartile range and the whiskers the 3 rd and 97 th percentile of the data. Significance testing was performed using paired Students T-test and values for p
    Edta, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 524 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/edta/product/Roche
    Average 99 stars, based on 524 article reviews
    Price from $9.99 to $1999.99
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    85
    Roche bj edta
    The interaction between HCV glycoproteins and CD81 is blocked when CD81 interacts with partner protein(s). A, HCV E1E2 heterodimers immobilized onto anti-E2 coated beads interacted with CD81 in all cells lysed in Triton X-100 (TX). However, the interaction of HCV-E1E2 with CD81 was blocked in Daudi and Ramos cells lysed in Brij (Bj). Maintenance of CD81 with other tetraspanins (T) and partners (P) is disrupted by the indicated lysis conditions, as diagrammed. Anti-CD81 and irrelevant (Cont) mAbs were used in immunoprecipitations as controls. Precipitation of CD81 was revealed by western blotting with the anti-CD81 5A6 mAb. CD81-LEL corresponds to the large extracellular loop of CD81 fused to the glutathione-S transferase. B, After cell surface biotinylation, the indicated cell lines were lysed with <t>Bj/EDTA,</t> immunoprecipitated with 5A6 mAb and the proteins revealed by Western blotting with HRP-conjugated streptavidin. The values on the right are molecular sizes in kilodaltons. C, EWI-2wint production is directly connected to EWI-2 expression. Daudi cells interfered with negative siRNA or EWI-2 siRNA were <t>biotinylated,</t> lysed in Bj/EDTA, immunoprecipitated with the anti-CD81 5A6 mAb or an anti-EWI-2 mAb (8A12) and blotted sequentially with HRP-conjugated streptavidin and 5A6 mAb.
    Bj Edta, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Roche apo state
    NMR spectra of SUPC2 Ccore under <t>holo</t> and <t>apo</t> conditions. Comparison of 1 H- 15 N HSQC NMR spectra of 13 C 15 N SUPC2 Ccore in the Ca 2+ -saturating condition (red contours) and in the Ca 2+ -free condition (green contours) in pH 7.4, 2 m m Tris-d 11 , 150 m m KCl buffer with 1 m m TCEP at 25 °C.
    Apo State, supplied by Roche, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cytokeratin 8 and CD3γ copy numbers in intestinal epithelial cells. A) FACS analysis of T cell contamination. Positive selection of IEC was performed using CD326 Microbeads followed by staining using T-cell receptor antibodies and Pacific Blue Annexin V to detect apoptotic cells. Panel A1 shows an example of IEC isolated using the enzyme based protocol, panel A2 using the EDTA/DTT based protocol. T-cell receptor positive cells are located in quadrant Q1 and Q2. Analysis of absolute mRNA copy numbers of cytokeratin 8 and CD3γ in intestinal epithelial cells separated by positive selection using CD326 Microbeads cells. B) Expression of mRNA copy numbers of Cytokeratin 8 and CD3γ. Data is expressed as box and whisker plots. The length of the boxes represents the interquartile range and the whiskers the 3 rd and 97 th percentile of the data. Significance testing was performed using paired Students T-test and values for p

    Journal: PLoS ONE

    Article Title: DNA Methylation Analysis in the Intestinal Epithelium--Effect of Cell Separation on Gene Expression and Methylation Profile

    doi: 10.1371/journal.pone.0055636

    Figure Lengend Snippet: Cytokeratin 8 and CD3γ copy numbers in intestinal epithelial cells. A) FACS analysis of T cell contamination. Positive selection of IEC was performed using CD326 Microbeads followed by staining using T-cell receptor antibodies and Pacific Blue Annexin V to detect apoptotic cells. Panel A1 shows an example of IEC isolated using the enzyme based protocol, panel A2 using the EDTA/DTT based protocol. T-cell receptor positive cells are located in quadrant Q1 and Q2. Analysis of absolute mRNA copy numbers of cytokeratin 8 and CD3γ in intestinal epithelial cells separated by positive selection using CD326 Microbeads cells. B) Expression of mRNA copy numbers of Cytokeratin 8 and CD3γ. Data is expressed as box and whisker plots. The length of the boxes represents the interquartile range and the whiskers the 3 rd and 97 th percentile of the data. Significance testing was performed using paired Students T-test and values for p

    Article Snippet: Four pooled biopsy samples were then subjected to either chemical digestion using a combination of EDTA and DTT, or enzymatic digestion with Liberase (a blend of collagenase I/II and a neutral protease; Roche Applied Science) and hyaluronidase.

    Techniques: FACS, Selection, Staining, Isolation, Expressing, Whisker Assay

    Influence of isolation methods on gene expression profiles. A) Differentially regulated genes in IEC isolated with EDTA/DTT compared to enzymatic release. Data is expressed as box and whisker plots. The length of the boxes represents the interquartile range and the whiskers the 3 rd and 97 th percentile of the data. Significance testing was performed using paired Students T-test and values for p

    Journal: PLoS ONE

    Article Title: DNA Methylation Analysis in the Intestinal Epithelium--Effect of Cell Separation on Gene Expression and Methylation Profile

    doi: 10.1371/journal.pone.0055636

    Figure Lengend Snippet: Influence of isolation methods on gene expression profiles. A) Differentially regulated genes in IEC isolated with EDTA/DTT compared to enzymatic release. Data is expressed as box and whisker plots. The length of the boxes represents the interquartile range and the whiskers the 3 rd and 97 th percentile of the data. Significance testing was performed using paired Students T-test and values for p

    Article Snippet: Four pooled biopsy samples were then subjected to either chemical digestion using a combination of EDTA and DTT, or enzymatic digestion with Liberase (a blend of collagenase I/II and a neutral protease; Roche Applied Science) and hyaluronidase.

    Techniques: Isolation, Expressing, Whisker Assay

    Quantitative PCR. Total RNA extracted from tibiae decalicified with 0.5 M EDTA, RNA later /EDTA at pH 9.2 or RNA later /EDTA at pH5.2 was analyzed for Col2a1 (A) or Rpl10 (B) mRNA transcripts by q PCR using Sybr Green. The machine output (Roche LightCycler 480 II) of fluorescence at each PCR cycle number is shown.

    Journal: PLoS ONE

    Article Title: Maintaining mRNA Integrity during Decalcification of Mineralized Tissues

    doi: 10.1371/journal.pone.0058154

    Figure Lengend Snippet: Quantitative PCR. Total RNA extracted from tibiae decalicified with 0.5 M EDTA, RNA later /EDTA at pH 9.2 or RNA later /EDTA at pH5.2 was analyzed for Col2a1 (A) or Rpl10 (B) mRNA transcripts by q PCR using Sybr Green. The machine output (Roche LightCycler 480 II) of fluorescence at each PCR cycle number is shown.

    Article Snippet: Quantitative PCR RNA isolated from EDTA or RNAlater /EDTA decalcified whole tibiae (500 ng total RNA) was reverse-transcribed as per the manufacturer’s protocol in a 20µL reaction using the Transcriptor High Fidelity cDNA Synthesis Kit (Roche, Germany).

    Techniques: Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, SYBR Green Assay, Fluorescence

    Fluorescent immunohistochemistry of articular cartilage. Collagen II (A, B) and collagen VI (C, D) protein localization was unaffected by RNA later /EDTA, pH 5.2 decalcification (B, D) compared to conventional EDTA decalcification (A, C). Using both methods collagen II can be seen in both the pericellular and extracellular matrix, while collagen VI is predominantly localized to the pericellular matrix. DAPI was used as a nuclear stain. Scale bars = 50 µm.

    Journal: PLoS ONE

    Article Title: Maintaining mRNA Integrity during Decalcification of Mineralized Tissues

    doi: 10.1371/journal.pone.0058154

    Figure Lengend Snippet: Fluorescent immunohistochemistry of articular cartilage. Collagen II (A, B) and collagen VI (C, D) protein localization was unaffected by RNA later /EDTA, pH 5.2 decalcification (B, D) compared to conventional EDTA decalcification (A, C). Using both methods collagen II can be seen in both the pericellular and extracellular matrix, while collagen VI is predominantly localized to the pericellular matrix. DAPI was used as a nuclear stain. Scale bars = 50 µm.

    Article Snippet: Quantitative PCR RNA isolated from EDTA or RNAlater /EDTA decalcified whole tibiae (500 ng total RNA) was reverse-transcribed as per the manufacturer’s protocol in a 20µL reaction using the Transcriptor High Fidelity cDNA Synthesis Kit (Roche, Germany).

    Techniques: Immunohistochemistry, Staining

    In situ hybridization for Prg4 and Col2a1 . Cryosections from tibia decalcified with EDTA (A,B) or RNA later /EDTA at pH5.2 (C–F) were hybridized with DIG-labeled Prg4 antisense (A,C) or sense (B,D) RNA probes, and against 35 S-labeled Col2a1 antisense (E) or sense (F) RNA probes for Col2a1 . Arrows show representative regions of target gene mRNA expression. Scale bars = 100 µm (A–D), 10 µm (E, F).

    Journal: PLoS ONE

    Article Title: Maintaining mRNA Integrity during Decalcification of Mineralized Tissues

    doi: 10.1371/journal.pone.0058154

    Figure Lengend Snippet: In situ hybridization for Prg4 and Col2a1 . Cryosections from tibia decalcified with EDTA (A,B) or RNA later /EDTA at pH5.2 (C–F) were hybridized with DIG-labeled Prg4 antisense (A,C) or sense (B,D) RNA probes, and against 35 S-labeled Col2a1 antisense (E) or sense (F) RNA probes for Col2a1 . Arrows show representative regions of target gene mRNA expression. Scale bars = 100 µm (A–D), 10 µm (E, F).

    Article Snippet: Quantitative PCR RNA isolated from EDTA or RNAlater /EDTA decalcified whole tibiae (500 ng total RNA) was reverse-transcribed as per the manufacturer’s protocol in a 20µL reaction using the Transcriptor High Fidelity cDNA Synthesis Kit (Roche, Germany).

    Techniques: In Situ Hybridization, Labeling, Expressing

    Cartilage morphology after EDTA or RNA later /EDTA decalcification. Tibial epiphyses were decalcified for 72 hrs at 4°C with 0.5 M EDTA (A) or RNA later /10% EDTA at pH 5.2 and cryosections were stained with toluidine blue/fast green. The medial tibial plateau is shown. Cartilage morphology and aggrecan staining is preserved in the RNA later /10% EDTA, pH 5.2 decalcified samples. Scale bar = 100 µm.

    Journal: PLoS ONE

    Article Title: Maintaining mRNA Integrity during Decalcification of Mineralized Tissues

    doi: 10.1371/journal.pone.0058154

    Figure Lengend Snippet: Cartilage morphology after EDTA or RNA later /EDTA decalcification. Tibial epiphyses were decalcified for 72 hrs at 4°C with 0.5 M EDTA (A) or RNA later /10% EDTA at pH 5.2 and cryosections were stained with toluidine blue/fast green. The medial tibial plateau is shown. Cartilage morphology and aggrecan staining is preserved in the RNA later /10% EDTA, pH 5.2 decalcified samples. Scale bar = 100 µm.

    Article Snippet: Quantitative PCR RNA isolated from EDTA or RNAlater /EDTA decalcified whole tibiae (500 ng total RNA) was reverse-transcribed as per the manufacturer’s protocol in a 20µL reaction using the Transcriptor High Fidelity cDNA Synthesis Kit (Roche, Germany).

    Techniques: Staining

    Analysis of RNA integrity. Microcapillary electrophoresis of total RNA isolated from whole tibial epiphysis (A, C, E) or cryosections of tibiae (B, D, F) decalcified with 0.5 M EDTA (A, B), RNA later /EDTA at pH 9.2 (C, D) or RNA later /EDTA at pH5.2 (E, F).

    Journal: PLoS ONE

    Article Title: Maintaining mRNA Integrity during Decalcification of Mineralized Tissues

    doi: 10.1371/journal.pone.0058154

    Figure Lengend Snippet: Analysis of RNA integrity. Microcapillary electrophoresis of total RNA isolated from whole tibial epiphysis (A, C, E) or cryosections of tibiae (B, D, F) decalcified with 0.5 M EDTA (A, B), RNA later /EDTA at pH 9.2 (C, D) or RNA later /EDTA at pH5.2 (E, F).

    Article Snippet: Quantitative PCR RNA isolated from EDTA or RNAlater /EDTA decalcified whole tibiae (500 ng total RNA) was reverse-transcribed as per the manufacturer’s protocol in a 20µL reaction using the Transcriptor High Fidelity cDNA Synthesis Kit (Roche, Germany).

    Techniques: Electrophoresis, Isolation

    The interaction between HCV glycoproteins and CD81 is blocked when CD81 interacts with partner protein(s). A, HCV E1E2 heterodimers immobilized onto anti-E2 coated beads interacted with CD81 in all cells lysed in Triton X-100 (TX). However, the interaction of HCV-E1E2 with CD81 was blocked in Daudi and Ramos cells lysed in Brij (Bj). Maintenance of CD81 with other tetraspanins (T) and partners (P) is disrupted by the indicated lysis conditions, as diagrammed. Anti-CD81 and irrelevant (Cont) mAbs were used in immunoprecipitations as controls. Precipitation of CD81 was revealed by western blotting with the anti-CD81 5A6 mAb. CD81-LEL corresponds to the large extracellular loop of CD81 fused to the glutathione-S transferase. B, After cell surface biotinylation, the indicated cell lines were lysed with Bj/EDTA, immunoprecipitated with 5A6 mAb and the proteins revealed by Western blotting with HRP-conjugated streptavidin. The values on the right are molecular sizes in kilodaltons. C, EWI-2wint production is directly connected to EWI-2 expression. Daudi cells interfered with negative siRNA or EWI-2 siRNA were biotinylated, lysed in Bj/EDTA, immunoprecipitated with the anti-CD81 5A6 mAb or an anti-EWI-2 mAb (8A12) and blotted sequentially with HRP-conjugated streptavidin and 5A6 mAb.

    Journal: PLoS ONE

    Article Title: The CD81 Partner EWI-2wint Inhibits Hepatitis C Virus Entry

    doi: 10.1371/journal.pone.0001866

    Figure Lengend Snippet: The interaction between HCV glycoproteins and CD81 is blocked when CD81 interacts with partner protein(s). A, HCV E1E2 heterodimers immobilized onto anti-E2 coated beads interacted with CD81 in all cells lysed in Triton X-100 (TX). However, the interaction of HCV-E1E2 with CD81 was blocked in Daudi and Ramos cells lysed in Brij (Bj). Maintenance of CD81 with other tetraspanins (T) and partners (P) is disrupted by the indicated lysis conditions, as diagrammed. Anti-CD81 and irrelevant (Cont) mAbs were used in immunoprecipitations as controls. Precipitation of CD81 was revealed by western blotting with the anti-CD81 5A6 mAb. CD81-LEL corresponds to the large extracellular loop of CD81 fused to the glutathione-S transferase. B, After cell surface biotinylation, the indicated cell lines were lysed with Bj/EDTA, immunoprecipitated with 5A6 mAb and the proteins revealed by Western blotting with HRP-conjugated streptavidin. The values on the right are molecular sizes in kilodaltons. C, EWI-2wint production is directly connected to EWI-2 expression. Daudi cells interfered with negative siRNA or EWI-2 siRNA were biotinylated, lysed in Bj/EDTA, immunoprecipitated with the anti-CD81 5A6 mAb or an anti-EWI-2 mAb (8A12) and blotted sequentially with HRP-conjugated streptavidin and 5A6 mAb.

    Article Snippet: Detection of cell surface biotinylated proteins Cells were biotinylated as previously described with 0.2 mg/ml EZ-link-Sulpho-NHS-LC-biotin (Pierce), and lysed into Bj/EDTA or TX/EDTA containing protease inhibitors (Complete, Roche).

    Techniques: Lysis, Western Blot, Immunoprecipitation, Expressing

    NMR spectra of SUPC2 Ccore under holo and apo conditions. Comparison of 1 H- 15 N HSQC NMR spectra of 13 C 15 N SUPC2 Ccore in the Ca 2+ -saturating condition (red contours) and in the Ca 2+ -free condition (green contours) in pH 7.4, 2 m m Tris-d 11 , 150 m m KCl buffer with 1 m m TCEP at 25 °C.

    Journal: The Journal of Biological Chemistry

    Article Title: Oligomerization of the Polycystin-2 C-terminal Tail and Effects on Its Ca2+-binding Properties *

    doi: 10.1074/jbc.M115.641803

    Figure Lengend Snippet: NMR spectra of SUPC2 Ccore under holo and apo conditions. Comparison of 1 H- 15 N HSQC NMR spectra of 13 C 15 N SUPC2 Ccore in the Ca 2+ -saturating condition (red contours) and in the Ca 2+ -free condition (green contours) in pH 7.4, 2 m m Tris-d 11 , 150 m m KCl buffer with 1 m m TCEP at 25 °C.

    Article Snippet: The NMR experiments were collected on 15 N- and 13 C-labeled 1.0 mm SUPC2 Ccore samples in both the holo state (20 mm CaCl2 ) and the apo state (1 mm EDTA) at pH 7.4 with 2 mm Tris-d 11 , 150 mm KCl, 1 mm TCEP, a protease inhibitor mixture (Roche Applied Science), and 5% (v/v) D2 O, with 5 mm sodium azide added as a preservative.

    Techniques: Nuclear Magnetic Resonance

    SEC-MALS results of HPC2 Cterm and SUPC2 Ccore in Ca 2+ -bound holo and Ca 2+ -free apo states. A , three different amounts of HPC2 Cterm protein were analyzed by Superdex 200 SEC-UV/LS/RI in Ca 2+ -saturating buffer (20 m m CaCl 2 ). The UV curves indicate the elution peaks of the protein samples analyzed, and the dotted lines indicate the calculated molar mass across the elution peaks. Orange , 1.16-mg injection; green , 155-μg injection; blue , 31.0-μg injection. B , three different amounts of HPC 2Cterm protein were analyzed by Superdex 200 SEC-UV/LS/RI in Ca 2+ -free buffer (no added CaCl 2 , 1 m m EDTA). Orange , 2.36-mg injection; green , 650-μg injection; blue , 260-μg injection. C , three different amounts of SUPC2 Ccore protein were analyzed by Superdex 200 SEC-UV/LS/RI in Ca 2+ -saturating buffer (20 m m CaCl 2 ). The UV curves indicate the elution peaks of the protein samples analyzed, and the dotted lines indicate the calculated molar mass across the elution peaks. Orange , 1.35-mg injection; green , 150-μg injection; blue , 15.0-μg injection. D , three different amounts of SUPC2 Ccore protein were analyzed by Superdex 200 SEC-UV/LS/RI in Ca 2+ -free buffer (no added CaCl 2 , 1 m m EDTA). Orange , 1.35-mg injection; green , 150-μg injection; blue , 75.2 μg injection.

    Journal: The Journal of Biological Chemistry

    Article Title: Oligomerization of the Polycystin-2 C-terminal Tail and Effects on Its Ca2+-binding Properties *

    doi: 10.1074/jbc.M115.641803

    Figure Lengend Snippet: SEC-MALS results of HPC2 Cterm and SUPC2 Ccore in Ca 2+ -bound holo and Ca 2+ -free apo states. A , three different amounts of HPC2 Cterm protein were analyzed by Superdex 200 SEC-UV/LS/RI in Ca 2+ -saturating buffer (20 m m CaCl 2 ). The UV curves indicate the elution peaks of the protein samples analyzed, and the dotted lines indicate the calculated molar mass across the elution peaks. Orange , 1.16-mg injection; green , 155-μg injection; blue , 31.0-μg injection. B , three different amounts of HPC 2Cterm protein were analyzed by Superdex 200 SEC-UV/LS/RI in Ca 2+ -free buffer (no added CaCl 2 , 1 m m EDTA). Orange , 2.36-mg injection; green , 650-μg injection; blue , 260-μg injection. C , three different amounts of SUPC2 Ccore protein were analyzed by Superdex 200 SEC-UV/LS/RI in Ca 2+ -saturating buffer (20 m m CaCl 2 ). The UV curves indicate the elution peaks of the protein samples analyzed, and the dotted lines indicate the calculated molar mass across the elution peaks. Orange , 1.35-mg injection; green , 150-μg injection; blue , 15.0-μg injection. D , three different amounts of SUPC2 Ccore protein were analyzed by Superdex 200 SEC-UV/LS/RI in Ca 2+ -free buffer (no added CaCl 2 , 1 m m EDTA). Orange , 1.35-mg injection; green , 150-μg injection; blue , 75.2 μg injection.

    Article Snippet: The NMR experiments were collected on 15 N- and 13 C-labeled 1.0 mm SUPC2 Ccore samples in both the holo state (20 mm CaCl2 ) and the apo state (1 mm EDTA) at pH 7.4 with 2 mm Tris-d 11 , 150 mm KCl, 1 mm TCEP, a protease inhibitor mixture (Roche Applied Science), and 5% (v/v) D2 O, with 5 mm sodium azide added as a preservative.

    Techniques: Size-exclusion Chromatography, Injection