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Roche protease inhibitor cocktail
Protease Inhibitor Cocktail, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 8944 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Clone Assay:

Article Title: 3.5? cryoEM Structure of Hepatitis B Virus Core Assembled from Full-Length Core Protein
Article Snippet: The amplified gene was double-digested with Nde I and Bam HI, and subsequently cloned into the expression vector pET-11a from Novagen. .. 60mg of chicken lysozyme, 15mg of DNAse1, and two tablets of Roche protease inhibitor cocktail were added into the cell suspension.

Centrifugation:

Article Title: Lysine Deacetylases Hda1 and Rpd3 Regulate Hsp90 Function thereby Governing Fungal Drug Resistance
Article Snippet: In brief, cells were washed with sterile water and resuspended in 500 μl of lysis buffer J containing 20 mM Tris pH 7.5, 100 mM KCl, 5 mM MgCl and 20% glycerol, with one protease inhibitor cocktail (complete, EDTA-free tablet, Roche Diagnostics) per 10 mL, as well as 1 mM PMSF (EMD Chemicals) and 20 mM sodium molybdate (Sigma Aldrich Co.) added fresh before use. .. Total collected lysates were cleared by centrifugation at 20817xg for 10 min at 4°C and protein concentrations were determined by Bradford analysis.

Article Title: 3.5? cryoEM Structure of Hepatitis B Virus Core Assembled from Full-Length Core Protein
Article Snippet: Bacterial cells were pelleted by 4000g centrifugation and then re-suspended in 75mL of PBS buffer. .. 60mg of chicken lysozyme, 15mg of DNAse1, and two tablets of Roche protease inhibitor cocktail were added into the cell suspension.

Article Title: Enhanced Discrimination of Malignant from Benign Pancreatic Disease by Measuring the CA 19-9 Antigen on Specific Protein Carriers
Article Snippet: The sandwich assay consisted of four 1-hour-incubations in room temperature (RT) with the following reagents: 1) blocking buffer (PBS containing 0.5% Tween-20 (PBST0.5) and 1% BSA); 2) a serum or plasma sample, diluted two-fold in 1×TBS containing 0.08% Brij, 0.08 Tween-20, 50 µg/ml protease inhibitor cocktail (Complete Protease Inhibitor Tablet, Roche Applied Science), and a cocktail of IgG from mouse, goat, and sheep each at 100 µg/ml and rabbit IgG at 200 µg/ml (Jackson ImmunoResearch Laboratories, Inc.); 3) biotinylated detection antibody or lectin (2 µg/ml), diluted in PBST0.1 containing 0.1% BSA; 4) streptavidin-phycoerythrin (10 µg/ml, Roche Applied Science), diluted in PBST0.1 containing 0.1% BSA. .. After each step, the slides were rinsed in three baths of PBST0.1 and dried by centrifugation (Eppendorf 5810R, rotor A-4-62, 1500× g).

Amplification:

Article Title: 3.5? cryoEM Structure of Hepatitis B Virus Core Assembled from Full-Length Core Protein
Article Snippet: The amplified gene was double-digested with Nde I and Bam HI, and subsequently cloned into the expression vector pET-11a from Novagen. .. 60mg of chicken lysozyme, 15mg of DNAse1, and two tablets of Roche protease inhibitor cocktail were added into the cell suspension.

Construct:

Article Title: Modified Cav1.4 Expression in the Cacna1fnob2 Mouse Due to Alternative Splicing of an ETn Inserted in Exon 2
Article Snippet: The resulting construct was transfected into tsA-201 cells (transfection details below). .. After a two-day incubation the cells were lysed on ice with lysis buffer (300 mM NaCl, 50 mM Tris pH 7.5, 0.1% Triton X-100) containing a protease-inhibitor cocktail (complete Mini EDTA-free, Roche).

Article Title: Distinct perinatal features of the hyperpolarization-activated non-selective cation current Ih in the rat cortical plate
Article Snippet: Tissue homogenization was performed in lysis buffer containing 20 mM Tris–HCl (pH 7.5), 0.25 M sucrose, 1 mM EGTA, and 5 mM EDTA which was supplemented with a protease inhibitor cocktail (PIC Complete, diluted according to the manufacturer; Roche Applied Science, Mannheim, Germany) and a phosphatase inhibitor cocktail (PhosSTOP, diluted according to the manufacturer; Roche). .. For protein extraction HEK293 cells were plated at a density of 20,000 cells/cm² (cultured as described above) and transfected (calcium phosphate) the next day with the constructs.

Microarray:

Article Title: Enhanced Discrimination of Malignant from Benign Pancreatic Disease by Measuring the CA 19-9 Antigen on Specific Protein Carriers
Article Snippet: Paragraph title: Microarray assays ... The sandwich assay consisted of four 1-hour-incubations in room temperature (RT) with the following reagents: 1) blocking buffer (PBS containing 0.5% Tween-20 (PBST0.5) and 1% BSA); 2) a serum or plasma sample, diluted two-fold in 1×TBS containing 0.08% Brij, 0.08 Tween-20, 50 µg/ml protease inhibitor cocktail (Complete Protease Inhibitor Tablet, Roche Applied Science), and a cocktail of IgG from mouse, goat, and sheep each at 100 µg/ml and rabbit IgG at 200 µg/ml (Jackson ImmunoResearch Laboratories, Inc.); 3) biotinylated detection antibody or lectin (2 µg/ml), diluted in PBST0.1 containing 0.1% BSA; 4) streptavidin-phycoerythrin (10 µg/ml, Roche Applied Science), diluted in PBST0.1 containing 0.1% BSA.

Incubation:

Article Title: Ezrin is a Major Regulator of Membrane Tension in Epithelial Cells
Article Snippet: .. After washing cells three times with PBS− , cell lysis was achieved by incubation with radioimmunoprecipitation (RIPA) buffer containing a protease inhibitor cocktail (cOmplete EDTA-free tablets; Roche Diagnostics, Mannheim, Germany) for 5 min at 0 °C. .. An adequate amount of the soluble fraction was diluted with SDS-PAGE sample buffer, shook at 85 °C for 5 min at 300 rpm and resolved by 17% SDS-PAGE.

Article Title: Functional differences of short and long isoforms of spastin harboring missense mutation
Article Snippet: Immunoblot assays, immunoprecipitation and densitometry analysis HeLa or HEK cells were lysed 18-24 h after transfection in TSE (50 mM Tris HCl pH 8.0, 150 mM NaCl, 1 mM EDTA), to which was added 1% Triton X-100 and protease inhibitor cocktail (Complete ULTRA Tablets, EDTA-Free, Roche) on ice. .. For immunoprecipitation experiments, 1 mg protein extract was incubated overnight in a rotator at 4°C with 50 µl Anti-Flag M2 magnetic beads (Sigma-Aldrich).

Article Title: Modified Cav1.4 Expression in the Cacna1fnob2 Mouse Due to Alternative Splicing of an ETn Inserted in Exon 2
Article Snippet: .. After a two-day incubation the cells were lysed on ice with lysis buffer (300 mM NaCl, 50 mM Tris pH 7.5, 0.1% Triton X-100) containing a protease-inhibitor cocktail (complete Mini EDTA-free, Roche). .. The lysate was incubated with sepharose G beads (Amersham) and Anti-HA High Affinity antibody (Roche) at 4°C for 4 hours.

Stripping Membranes:

Article Title: Reduced insulin action in muscle of high fat diet rats over the diurnal cycle is not associated with defective insulin signaling
Article Snippet: 2.8 Immunoblotting Frozen, powdered tissue was homogenized by probe homogenizer in RIPA buffer containing protease inhibitor cocktail (1 × EDTA free; Roche) and phosphatase inhibitors (10 mM NaF, 1 mM Na3 VO4 ). .. Total amount of each protein was quantified on the same membranes as phospho-proteins after stripping (0.5 M NaOH, 30 min) and reprobing.

Expressing:

Article Title: 3.5? cryoEM Structure of Hepatitis B Virus Core Assembled from Full-Length Core Protein
Article Snippet: Paragraph title: Protein expression and HBV core purification ... 60mg of chicken lysozyme, 15mg of DNAse1, and two tablets of Roche protease inhibitor cocktail were added into the cell suspension.

Article Title: Modified Cav1.4 Expression in the Cacna1fnob2 Mouse Due to Alternative Splicing of an ETn Inserted in Exon 2
Article Snippet: Each fragment was ligated into the T-easy vector system (Promega), the sequence was confirmed, and the fragment was subcloned into the expression vector pCDNA3.1neo. .. After a two-day incubation the cells were lysed on ice with lysis buffer (300 mM NaCl, 50 mM Tris pH 7.5, 0.1% Triton X-100) containing a protease-inhibitor cocktail (complete Mini EDTA-free, Roche).

Article Title: Characterization and Modeling of the Oligomeric State and Ligand Binding Behavior of Purified Translocator Protein 18 kDa from Rhodobacter sphaeroides
Article Snippet: Paragraph title: Protein Expression and Purification ... Harvested cells were resuspended in lysis buffer [50 mM KH2 PO4 (pH 6.5) and 1 mM EDTA] supplemented with Roche protease inhibitor cocktail and lysed via two passes at 20000 psi through a French press homogenizer.

Western Blot:

Article Title: Ezrin is a Major Regulator of Membrane Tension in Epithelial Cells
Article Snippet: Paragraph title: Solubilisation, Purification and Western Blot Analysis ... After washing cells three times with PBS− , cell lysis was achieved by incubation with radioimmunoprecipitation (RIPA) buffer containing a protease inhibitor cocktail (cOmplete EDTA-free tablets; Roche Diagnostics, Mannheim, Germany) for 5 min at 0 °C.

Article Title: Cisplatin and Doxorubicin Induce Distinct Mechanisms of Ovarian Follicle Loss; Imatinib Provides Selective Protection Only against Cisplatin
Article Snippet: .. Protein Extraction and Western Blotting Frozen ovaries were homogenised in 20 µl lysis buffer (containing 50 mM HEPES buffer, 10% Triton X, 50 mM NaCl, Protease inhibitor cocktail and protease inhibitors [I and II], H2 O; all purchased from Sigma Aldrich Ltd except for Protease inhibitor cocktail which was purchased from Roche Diagnostic Ltd) and centrifuged at 13000 rpm for 20 mins: supernatant was used for Western blotting. ..

Transformation Assay:

Article Title: 3.5? cryoEM Structure of Hepatitis B Virus Core Assembled from Full-Length Core Protein
Article Snippet: Transformed BL21 (DE3) cells were grown in LB medium with 100µg/ml of ampicillin at 37°C overnight. .. 60mg of chicken lysozyme, 15mg of DNAse1, and two tablets of Roche protease inhibitor cocktail were added into the cell suspension.

Transfection:

Article Title: Functional differences of short and long isoforms of spastin harboring missense mutation
Article Snippet: .. Immunoblot assays, immunoprecipitation and densitometry analysis HeLa or HEK cells were lysed 18-24 h after transfection in TSE (50 mM Tris HCl pH 8.0, 150 mM NaCl, 1 mM EDTA), to which was added 1% Triton X-100 and protease inhibitor cocktail (Complete ULTRA Tablets, EDTA-Free, Roche) on ice. .. For immunoprecipitation experiments, 1 mg protein extract was incubated overnight in a rotator at 4°C with 50 µl Anti-Flag M2 magnetic beads (Sigma-Aldrich).

Article Title: Modified Cav1.4 Expression in the Cacna1fnob2 Mouse Due to Alternative Splicing of an ETn Inserted in Exon 2
Article Snippet: The resulting construct was transfected into tsA-201 cells (transfection details below). .. After a two-day incubation the cells were lysed on ice with lysis buffer (300 mM NaCl, 50 mM Tris pH 7.5, 0.1% Triton X-100) containing a protease-inhibitor cocktail (complete Mini EDTA-free, Roche).

Article Title: Distinct perinatal features of the hyperpolarization-activated non-selective cation current Ih in the rat cortical plate
Article Snippet: Tissue homogenization was performed in lysis buffer containing 20 mM Tris–HCl (pH 7.5), 0.25 M sucrose, 1 mM EGTA, and 5 mM EDTA which was supplemented with a protease inhibitor cocktail (PIC Complete, diluted according to the manufacturer; Roche Applied Science, Mannheim, Germany) and a phosphatase inhibitor cocktail (PhosSTOP, diluted according to the manufacturer; Roche). .. For protein extraction HEK293 cells were plated at a density of 20,000 cells/cm² (cultured as described above) and transfected (calcium phosphate) the next day with the constructs.

Immunoprecipitation:

Article Title: Functional differences of short and long isoforms of spastin harboring missense mutation
Article Snippet: .. Immunoblot assays, immunoprecipitation and densitometry analysis HeLa or HEK cells were lysed 18-24 h after transfection in TSE (50 mM Tris HCl pH 8.0, 150 mM NaCl, 1 mM EDTA), to which was added 1% Triton X-100 and protease inhibitor cocktail (Complete ULTRA Tablets, EDTA-Free, Roche) on ice. .. For immunoprecipitation experiments, 1 mg protein extract was incubated overnight in a rotator at 4°C with 50 µl Anti-Flag M2 magnetic beads (Sigma-Aldrich).

Article Title: Lysine Deacetylases Hda1 and Rpd3 Regulate Hsp90 Function thereby Governing Fungal Drug Resistance
Article Snippet: Paragraph title: Immunoprecipitation ... In brief, cells were washed with sterile water and resuspended in 500 μl of lysis buffer J containing 20 mM Tris pH 7.5, 100 mM KCl, 5 mM MgCl and 20% glycerol, with one protease inhibitor cocktail (complete, EDTA-free tablet, Roche Diagnostics) per 10 mL, as well as 1 mM PMSF (EMD Chemicals) and 20 mM sodium molybdate (Sigma Aldrich Co.) added fresh before use.

Protease Inhibitor:

Article Title: Ezrin is a Major Regulator of Membrane Tension in Epithelial Cells
Article Snippet: .. After washing cells three times with PBS− , cell lysis was achieved by incubation with radioimmunoprecipitation (RIPA) buffer containing a protease inhibitor cocktail (cOmplete EDTA-free tablets; Roche Diagnostics, Mannheim, Germany) for 5 min at 0 °C. .. An adequate amount of the soluble fraction was diluted with SDS-PAGE sample buffer, shook at 85 °C for 5 min at 300 rpm and resolved by 17% SDS-PAGE.

Article Title: Functional differences of short and long isoforms of spastin harboring missense mutation
Article Snippet: .. Immunoblot assays, immunoprecipitation and densitometry analysis HeLa or HEK cells were lysed 18-24 h after transfection in TSE (50 mM Tris HCl pH 8.0, 150 mM NaCl, 1 mM EDTA), to which was added 1% Triton X-100 and protease inhibitor cocktail (Complete ULTRA Tablets, EDTA-Free, Roche) on ice. .. For immunoprecipitation experiments, 1 mg protein extract was incubated overnight in a rotator at 4°C with 50 µl Anti-Flag M2 magnetic beads (Sigma-Aldrich).

Article Title: Cisplatin and Doxorubicin Induce Distinct Mechanisms of Ovarian Follicle Loss; Imatinib Provides Selective Protection Only against Cisplatin
Article Snippet: .. Protein Extraction and Western Blotting Frozen ovaries were homogenised in 20 µl lysis buffer (containing 50 mM HEPES buffer, 10% Triton X, 50 mM NaCl, Protease inhibitor cocktail and protease inhibitors [I and II], H2 O; all purchased from Sigma Aldrich Ltd except for Protease inhibitor cocktail which was purchased from Roche Diagnostic Ltd) and centrifuged at 13000 rpm for 20 mins: supernatant was used for Western blotting. ..

Article Title: Lysine Deacetylases Hda1 and Rpd3 Regulate Hsp90 Function thereby Governing Fungal Drug Resistance
Article Snippet: .. In brief, cells were washed with sterile water and resuspended in 500 μl of lysis buffer J containing 20 mM Tris pH 7.5, 100 mM KCl, 5 mM MgCl and 20% glycerol, with one protease inhibitor cocktail (complete, EDTA-free tablet, Roche Diagnostics) per 10 mL, as well as 1 mM PMSF (EMD Chemicals) and 20 mM sodium molybdate (Sigma Aldrich Co.) added fresh before use. ..

Article Title: Reduced insulin action in muscle of high fat diet rats over the diurnal cycle is not associated with defective insulin signaling
Article Snippet: .. 2.8 Immunoblotting Frozen, powdered tissue was homogenized by probe homogenizer in RIPA buffer containing protease inhibitor cocktail (1 × EDTA free; Roche) and phosphatase inhibitors (10 mM NaF, 1 mM Na3 VO4 ). .. Tissue lysates were subjected to SDS-PAGE, transferred to PVDF membranes, blocked in 2% BSA and then immunoblotted with antibodies for pAkt Ser 473 (#9271), pan Akt (#9272), pAS160 Thr642 (#4288), AS160 (#2670) pGSK3β Ser9 (#9336) and GSK3β (#9315) (Cell Signaling Technology, Beverly, USA).

Article Title: Identification of Ocular Autoantigens Associated With Juvenile Idiopathic Arthritis-Associated Uveitis
Article Snippet: .. The tissue specimens were then homogenized in urea lysis buffer (9 M urea, 2 M thiourea, 65 mM CHAPS, and 65 mM DTT) supplemented with 1 mM PMSF (SERVA, Heidelberg, Germany) and protease inhibitor cocktail (Complete, EDTA-free protease inhibitor cocktail, Roche Diagnostics, Mannheim, Germany) using ReadyPrep Mini Grinders according to the manufacturer's instructions (Bio-Rad, Munich, Germany). ..

Article Title: 3.5? cryoEM Structure of Hepatitis B Virus Core Assembled from Full-Length Core Protein
Article Snippet: .. 60mg of chicken lysozyme, 15mg of DNAse1, and two tablets of Roche protease inhibitor cocktail were added into the cell suspension. ..

Article Title: Modified Cav1.4 Expression in the Cacna1fnob2 Mouse Due to Alternative Splicing of an ETn Inserted in Exon 2
Article Snippet: .. After a two-day incubation the cells were lysed on ice with lysis buffer (300 mM NaCl, 50 mM Tris pH 7.5, 0.1% Triton X-100) containing a protease-inhibitor cocktail (complete Mini EDTA-free, Roche). .. The lysate was incubated with sepharose G beads (Amersham) and Anti-HA High Affinity antibody (Roche) at 4°C for 4 hours.

Article Title: Characterization and Modeling of the Oligomeric State and Ligand Binding Behavior of Purified Translocator Protein 18 kDa from Rhodobacter sphaeroides
Article Snippet: .. Harvested cells were resuspended in lysis buffer [50 mM KH2 PO4 (pH 6.5) and 1 mM EDTA] supplemented with Roche protease inhibitor cocktail and lysed via two passes at 20000 psi through a French press homogenizer. ..

Article Title: Distinct perinatal features of the hyperpolarization-activated non-selective cation current Ih in the rat cortical plate
Article Snippet: .. Tissue homogenization was performed in lysis buffer containing 20 mM Tris–HCl (pH 7.5), 0.25 M sucrose, 1 mM EGTA, and 5 mM EDTA which was supplemented with a protease inhibitor cocktail (PIC Complete, diluted according to the manufacturer; Roche Applied Science, Mannheim, Germany) and a phosphatase inhibitor cocktail (PhosSTOP, diluted according to the manufacturer; Roche). .. As positive controls we used protein extracts from HEK293 cells over-expressing either pIRES2-dsRed-rHCN1 [ ], pIRES2-eGFP-rHCN2 (kindly provided by Shigetada Nakanishi), rHCN3-pcDNA1 (kindly provided by Prof. Müller, Research Centre Jülich), or hHCN4-pcDNA3 (kindly provided by Juliane Stieber, University of Erlangen).

Article Title: Enhanced Discrimination of Malignant from Benign Pancreatic Disease by Measuring the CA 19-9 Antigen on Specific Protein Carriers
Article Snippet: .. The sandwich assay consisted of four 1-hour-incubations in room temperature (RT) with the following reagents: 1) blocking buffer (PBS containing 0.5% Tween-20 (PBST0.5) and 1% BSA); 2) a serum or plasma sample, diluted two-fold in 1×TBS containing 0.08% Brij, 0.08 Tween-20, 50 µg/ml protease inhibitor cocktail (Complete Protease Inhibitor Tablet, Roche Applied Science), and a cocktail of IgG from mouse, goat, and sheep each at 100 µg/ml and rabbit IgG at 200 µg/ml (Jackson ImmunoResearch Laboratories, Inc.); 3) biotinylated detection antibody or lectin (2 µg/ml), diluted in PBST0.1 containing 0.1% BSA; 4) streptavidin-phycoerythrin (10 µg/ml, Roche Applied Science), diluted in PBST0.1 containing 0.1% BSA. .. After each step, the slides were rinsed in three baths of PBST0.1 and dried by centrifugation (Eppendorf 5810R, rotor A-4-62, 1500× g).

Article Title: Oesophagostomum dentatum Extract Modulates T Cell-Dependent Immune Responses to Bystander Antigens and Prevents the Development of Allergy in Mice
Article Snippet: .. Bronchoalvelar Lavage and Differential Cell Counts Bronchoalveolar lavage fluid (BALF) was collected by cannulating the trachea and injecting/recovering 2×1 ml PBS containing a protease inhibitor cocktail (Complete® Protease Inhibitor cocktail Tablets; Roche, Mannheim, Germany). ..

Cell Culture:

Article Title: Ezrin is a Major Regulator of Membrane Tension in Epithelial Cells
Article Snippet: Solubilisation, Purification and Western Blot Analysis Cells were grown to confluency in 6-well cell culture plates. .. After washing cells three times with PBS− , cell lysis was achieved by incubation with radioimmunoprecipitation (RIPA) buffer containing a protease inhibitor cocktail (cOmplete EDTA-free tablets; Roche Diagnostics, Mannheim, Germany) for 5 min at 0 °C.

Article Title: Distinct perinatal features of the hyperpolarization-activated non-selective cation current Ih in the rat cortical plate
Article Snippet: Tissue homogenization was performed in lysis buffer containing 20 mM Tris–HCl (pH 7.5), 0.25 M sucrose, 1 mM EGTA, and 5 mM EDTA which was supplemented with a protease inhibitor cocktail (PIC Complete, diluted according to the manufacturer; Roche Applied Science, Mannheim, Germany) and a phosphatase inhibitor cocktail (PhosSTOP, diluted according to the manufacturer; Roche). .. For protein extraction HEK293 cells were plated at a density of 20,000 cells/cm² (cultured as described above) and transfected (calcium phosphate) the next day with the constructs.

Protein Concentration:

Article Title: Characterization and Modeling of the Oligomeric State and Ligand Binding Behavior of Purified Translocator Protein 18 kDa from Rhodobacter sphaeroides
Article Snippet: Harvested cells were resuspended in lysis buffer [50 mM KH2 PO4 (pH 6.5) and 1 mM EDTA] supplemented with Roche protease inhibitor cocktail and lysed via two passes at 20000 psi through a French press homogenizer. .. Membranes were thawed on ice, resuspended to a total protein concentration of 10 mg/mL in buffer A, solubilized via addition of 1% DDM, 1 mM PMSF, and Roche protease inhibitor cocktail (no EDTA), and stirred at 4 °C for 1 h. Solubilized membranes were centrifuged at 150000g for 30 min to remove the unsolubilized fraction.

Polymerase Chain Reaction:

Article Title: Modified Cav1.4 Expression in the Cacna1fnob2 Mouse Due to Alternative Splicing of an ETn Inserted in Exon 2
Article Snippet: PCR was used to amplify (past repeat 23) and add an HA tag onto the C-terminal regions of filamins A and B. .. After a two-day incubation the cells were lysed on ice with lysis buffer (300 mM NaCl, 50 mM Tris pH 7.5, 0.1% Triton X-100) containing a protease-inhibitor cocktail (complete Mini EDTA-free, Roche).

Binding Assay:

Article Title: Enhanced Discrimination of Malignant from Benign Pancreatic Disease by Measuring the CA 19-9 Antigen on Specific Protein Carriers
Article Snippet: The sandwich assay consisted of four 1-hour-incubations in room temperature (RT) with the following reagents: 1) blocking buffer (PBS containing 0.5% Tween-20 (PBST0.5) and 1% BSA); 2) a serum or plasma sample, diluted two-fold in 1×TBS containing 0.08% Brij, 0.08 Tween-20, 50 µg/ml protease inhibitor cocktail (Complete Protease Inhibitor Tablet, Roche Applied Science), and a cocktail of IgG from mouse, goat, and sheep each at 100 µg/ml and rabbit IgG at 200 µg/ml (Jackson ImmunoResearch Laboratories, Inc.); 3) biotinylated detection antibody or lectin (2 µg/ml), diluted in PBST0.1 containing 0.1% BSA; 4) streptavidin-phycoerythrin (10 µg/ml, Roche Applied Science), diluted in PBST0.1 containing 0.1% BSA. .. The measurement of glycans by using lectins detection on the captured proteins ( ) was carried out as above, except the glycans on the spotted antibodies were derivatized to prevent lectin binding to the antibodies , and the arrays were probed with glycan-binding lectins.

Fluorescence:

Article Title: Enhanced Discrimination of Malignant from Benign Pancreatic Disease by Measuring the CA 19-9 Antigen on Specific Protein Carriers
Article Snippet: The sandwich assay consisted of four 1-hour-incubations in room temperature (RT) with the following reagents: 1) blocking buffer (PBS containing 0.5% Tween-20 (PBST0.5) and 1% BSA); 2) a serum or plasma sample, diluted two-fold in 1×TBS containing 0.08% Brij, 0.08 Tween-20, 50 µg/ml protease inhibitor cocktail (Complete Protease Inhibitor Tablet, Roche Applied Science), and a cocktail of IgG from mouse, goat, and sheep each at 100 µg/ml and rabbit IgG at 200 µg/ml (Jackson ImmunoResearch Laboratories, Inc.); 3) biotinylated detection antibody or lectin (2 µg/ml), diluted in PBST0.1 containing 0.1% BSA; 4) streptavidin-phycoerythrin (10 µg/ml, Roche Applied Science), diluted in PBST0.1 containing 0.1% BSA. .. Fluorescence emission from the phycoerythrin was detected at 570 nm using a microarray scanner (LS Reloaded, Tecan).

Magnetic Beads:

Article Title: Functional differences of short and long isoforms of spastin harboring missense mutation
Article Snippet: Immunoblot assays, immunoprecipitation and densitometry analysis HeLa or HEK cells were lysed 18-24 h after transfection in TSE (50 mM Tris HCl pH 8.0, 150 mM NaCl, 1 mM EDTA), to which was added 1% Triton X-100 and protease inhibitor cocktail (Complete ULTRA Tablets, EDTA-Free, Roche) on ice. .. For immunoprecipitation experiments, 1 mg protein extract was incubated overnight in a rotator at 4°C with 50 µl Anti-Flag M2 magnetic beads (Sigma-Aldrich).

Isolation:

Article Title: Identification of Ocular Autoantigens Associated With Juvenile Idiopathic Arthritis-Associated Uveitis
Article Snippet: Paragraph title: Protein Isolation ... The tissue specimens were then homogenized in urea lysis buffer (9 M urea, 2 M thiourea, 65 mM CHAPS, and 65 mM DTT) supplemented with 1 mM PMSF (SERVA, Heidelberg, Germany) and protease inhibitor cocktail (Complete, EDTA-free protease inhibitor cocktail, Roche Diagnostics, Mannheim, Germany) using ReadyPrep Mini Grinders according to the manufacturer's instructions (Bio-Rad, Munich, Germany).

Article Title: Characterization and Modeling of the Oligomeric State and Ligand Binding Behavior of Purified Translocator Protein 18 kDa from Rhodobacter sphaeroides
Article Snippet: Harvested cells were resuspended in lysis buffer [50 mM KH2 PO4 (pH 6.5) and 1 mM EDTA] supplemented with Roche protease inhibitor cocktail and lysed via two passes at 20000 psi through a French press homogenizer. .. Isolated membranes were resuspended with buffer A [50 mM Tris-HCl (pH 8), 150 mM NaCl, and 10% glycerol], quick frozen with liquid nitrogen, and stored at −70 °C before being purified.

Purification:

Article Title: Ezrin is a Major Regulator of Membrane Tension in Epithelial Cells
Article Snippet: Paragraph title: Solubilisation, Purification and Western Blot Analysis ... After washing cells three times with PBS− , cell lysis was achieved by incubation with radioimmunoprecipitation (RIPA) buffer containing a protease inhibitor cocktail (cOmplete EDTA-free tablets; Roche Diagnostics, Mannheim, Germany) for 5 min at 0 °C.

Article Title: 3.5? cryoEM Structure of Hepatitis B Virus Core Assembled from Full-Length Core Protein
Article Snippet: Paragraph title: Protein expression and HBV core purification ... 60mg of chicken lysozyme, 15mg of DNAse1, and two tablets of Roche protease inhibitor cocktail were added into the cell suspension.

Article Title: Characterization and Modeling of the Oligomeric State and Ligand Binding Behavior of Purified Translocator Protein 18 kDa from Rhodobacter sphaeroides
Article Snippet: Paragraph title: Protein Expression and Purification ... Harvested cells were resuspended in lysis buffer [50 mM KH2 PO4 (pH 6.5) and 1 mM EDTA] supplemented with Roche protease inhibitor cocktail and lysed via two passes at 20000 psi through a French press homogenizer.

Sequencing:

Article Title: Modified Cav1.4 Expression in the Cacna1fnob2 Mouse Due to Alternative Splicing of an ETn Inserted in Exon 2
Article Snippet: Each fragment was ligated into the T-easy vector system (Promega), the sequence was confirmed, and the fragment was subcloned into the expression vector pCDNA3.1neo. .. After a two-day incubation the cells were lysed on ice with lysis buffer (300 mM NaCl, 50 mM Tris pH 7.5, 0.1% Triton X-100) containing a protease-inhibitor cocktail (complete Mini EDTA-free, Roche).

Protein Extraction:

Article Title: Cisplatin and Doxorubicin Induce Distinct Mechanisms of Ovarian Follicle Loss; Imatinib Provides Selective Protection Only against Cisplatin
Article Snippet: .. Protein Extraction and Western Blotting Frozen ovaries were homogenised in 20 µl lysis buffer (containing 50 mM HEPES buffer, 10% Triton X, 50 mM NaCl, Protease inhibitor cocktail and protease inhibitors [I and II], H2 O; all purchased from Sigma Aldrich Ltd except for Protease inhibitor cocktail which was purchased from Roche Diagnostic Ltd) and centrifuged at 13000 rpm for 20 mins: supernatant was used for Western blotting. ..

Article Title: Distinct perinatal features of the hyperpolarization-activated non-selective cation current Ih in the rat cortical plate
Article Snippet: Tissue homogenization was performed in lysis buffer containing 20 mM Tris–HCl (pH 7.5), 0.25 M sucrose, 1 mM EGTA, and 5 mM EDTA which was supplemented with a protease inhibitor cocktail (PIC Complete, diluted according to the manufacturer; Roche Applied Science, Mannheim, Germany) and a phosphatase inhibitor cocktail (PhosSTOP, diluted according to the manufacturer; Roche). .. For protein extraction HEK293 cells were plated at a density of 20,000 cells/cm² (cultured as described above) and transfected (calcium phosphate) the next day with the constructs.

Blocking Assay:

Article Title: Enhanced Discrimination of Malignant from Benign Pancreatic Disease by Measuring the CA 19-9 Antigen on Specific Protein Carriers
Article Snippet: .. The sandwich assay consisted of four 1-hour-incubations in room temperature (RT) with the following reagents: 1) blocking buffer (PBS containing 0.5% Tween-20 (PBST0.5) and 1% BSA); 2) a serum or plasma sample, diluted two-fold in 1×TBS containing 0.08% Brij, 0.08 Tween-20, 50 µg/ml protease inhibitor cocktail (Complete Protease Inhibitor Tablet, Roche Applied Science), and a cocktail of IgG from mouse, goat, and sheep each at 100 µg/ml and rabbit IgG at 200 µg/ml (Jackson ImmunoResearch Laboratories, Inc.); 3) biotinylated detection antibody or lectin (2 µg/ml), diluted in PBST0.1 containing 0.1% BSA; 4) streptavidin-phycoerythrin (10 µg/ml, Roche Applied Science), diluted in PBST0.1 containing 0.1% BSA. .. After each step, the slides were rinsed in three baths of PBST0.1 and dried by centrifugation (Eppendorf 5810R, rotor A-4-62, 1500× g).

Polyacrylamide Gel Electrophoresis:

Article Title: Modified Cav1.4 Expression in the Cacna1fnob2 Mouse Due to Alternative Splicing of an ETn Inserted in Exon 2
Article Snippet: After a two-day incubation the cells were lysed on ice with lysis buffer (300 mM NaCl, 50 mM Tris pH 7.5, 0.1% Triton X-100) containing a protease-inhibitor cocktail (complete Mini EDTA-free, Roche). .. The beads were washed again, and then proteins were resolved using PAGE (12% gel), transferred to a PVDF membrane, and probed with Anti-GST antibody (1∶2000 dilution, GE Healthcare).

Lysis:

Article Title: Ezrin is a Major Regulator of Membrane Tension in Epithelial Cells
Article Snippet: .. After washing cells three times with PBS− , cell lysis was achieved by incubation with radioimmunoprecipitation (RIPA) buffer containing a protease inhibitor cocktail (cOmplete EDTA-free tablets; Roche Diagnostics, Mannheim, Germany) for 5 min at 0 °C. .. An adequate amount of the soluble fraction was diluted with SDS-PAGE sample buffer, shook at 85 °C for 5 min at 300 rpm and resolved by 17% SDS-PAGE.

Article Title: Cisplatin and Doxorubicin Induce Distinct Mechanisms of Ovarian Follicle Loss; Imatinib Provides Selective Protection Only against Cisplatin
Article Snippet: .. Protein Extraction and Western Blotting Frozen ovaries were homogenised in 20 µl lysis buffer (containing 50 mM HEPES buffer, 10% Triton X, 50 mM NaCl, Protease inhibitor cocktail and protease inhibitors [I and II], H2 O; all purchased from Sigma Aldrich Ltd except for Protease inhibitor cocktail which was purchased from Roche Diagnostic Ltd) and centrifuged at 13000 rpm for 20 mins: supernatant was used for Western blotting. ..

Article Title: Lysine Deacetylases Hda1 and Rpd3 Regulate Hsp90 Function thereby Governing Fungal Drug Resistance
Article Snippet: .. In brief, cells were washed with sterile water and resuspended in 500 μl of lysis buffer J containing 20 mM Tris pH 7.5, 100 mM KCl, 5 mM MgCl and 20% glycerol, with one protease inhibitor cocktail (complete, EDTA-free tablet, Roche Diagnostics) per 10 mL, as well as 1 mM PMSF (EMD Chemicals) and 20 mM sodium molybdate (Sigma Aldrich Co.) added fresh before use. ..

Article Title: Identification of Ocular Autoantigens Associated With Juvenile Idiopathic Arthritis-Associated Uveitis
Article Snippet: .. The tissue specimens were then homogenized in urea lysis buffer (9 M urea, 2 M thiourea, 65 mM CHAPS, and 65 mM DTT) supplemented with 1 mM PMSF (SERVA, Heidelberg, Germany) and protease inhibitor cocktail (Complete, EDTA-free protease inhibitor cocktail, Roche Diagnostics, Mannheim, Germany) using ReadyPrep Mini Grinders according to the manufacturer's instructions (Bio-Rad, Munich, Germany). ..

Article Title: Modified Cav1.4 Expression in the Cacna1fnob2 Mouse Due to Alternative Splicing of an ETn Inserted in Exon 2
Article Snippet: .. After a two-day incubation the cells were lysed on ice with lysis buffer (300 mM NaCl, 50 mM Tris pH 7.5, 0.1% Triton X-100) containing a protease-inhibitor cocktail (complete Mini EDTA-free, Roche). .. The lysate was incubated with sepharose G beads (Amersham) and Anti-HA High Affinity antibody (Roche) at 4°C for 4 hours.

Article Title: Characterization and Modeling of the Oligomeric State and Ligand Binding Behavior of Purified Translocator Protein 18 kDa from Rhodobacter sphaeroides
Article Snippet: .. Harvested cells were resuspended in lysis buffer [50 mM KH2 PO4 (pH 6.5) and 1 mM EDTA] supplemented with Roche protease inhibitor cocktail and lysed via two passes at 20000 psi through a French press homogenizer. ..

Article Title: Distinct perinatal features of the hyperpolarization-activated non-selective cation current Ih in the rat cortical plate
Article Snippet: .. Tissue homogenization was performed in lysis buffer containing 20 mM Tris–HCl (pH 7.5), 0.25 M sucrose, 1 mM EGTA, and 5 mM EDTA which was supplemented with a protease inhibitor cocktail (PIC Complete, diluted according to the manufacturer; Roche Applied Science, Mannheim, Germany) and a phosphatase inhibitor cocktail (PhosSTOP, diluted according to the manufacturer; Roche). .. As positive controls we used protein extracts from HEK293 cells over-expressing either pIRES2-dsRed-rHCN1 [ ], pIRES2-eGFP-rHCN2 (kindly provided by Shigetada Nakanishi), rHCN3-pcDNA1 (kindly provided by Prof. Müller, Research Centre Jülich), or hHCN4-pcDNA3 (kindly provided by Juliane Stieber, University of Erlangen).

SDS Page:

Article Title: Ezrin is a Major Regulator of Membrane Tension in Epithelial Cells
Article Snippet: After washing cells three times with PBS− , cell lysis was achieved by incubation with radioimmunoprecipitation (RIPA) buffer containing a protease inhibitor cocktail (cOmplete EDTA-free tablets; Roche Diagnostics, Mannheim, Germany) for 5 min at 0 °C. .. An adequate amount of the soluble fraction was diluted with SDS-PAGE sample buffer, shook at 85 °C for 5 min at 300 rpm and resolved by 17% SDS-PAGE.

Article Title: Functional differences of short and long isoforms of spastin harboring missense mutation
Article Snippet: Immunoblot assays, immunoprecipitation and densitometry analysis HeLa or HEK cells were lysed 18-24 h after transfection in TSE (50 mM Tris HCl pH 8.0, 150 mM NaCl, 1 mM EDTA), to which was added 1% Triton X-100 and protease inhibitor cocktail (Complete ULTRA Tablets, EDTA-Free, Roche) on ice. .. Between 40 µg and 60 µg lysates were separated by SDS-PAGE by using Novex Wedgewell 4-20% Tris Glycine (Invitrogen), and then the proteins were transferred onto nitrocellulose membrane (Amersham Protran, GE Healthcare Life Sciences).

Article Title: Reduced insulin action in muscle of high fat diet rats over the diurnal cycle is not associated with defective insulin signaling
Article Snippet: 2.8 Immunoblotting Frozen, powdered tissue was homogenized by probe homogenizer in RIPA buffer containing protease inhibitor cocktail (1 × EDTA free; Roche) and phosphatase inhibitors (10 mM NaF, 1 mM Na3 VO4 ). .. Tissue lysates were subjected to SDS-PAGE, transferred to PVDF membranes, blocked in 2% BSA and then immunoblotted with antibodies for pAkt Ser 473 (#9271), pan Akt (#9272), pAS160 Thr642 (#4288), AS160 (#2670) pGSK3β Ser9 (#9336) and GSK3β (#9315) (Cell Signaling Technology, Beverly, USA).

Plasmid Preparation:

Article Title: 3.5? cryoEM Structure of Hepatitis B Virus Core Assembled from Full-Length Core Protein
Article Snippet: The amplified gene was double-digested with Nde I and Bam HI, and subsequently cloned into the expression vector pET-11a from Novagen. .. 60mg of chicken lysozyme, 15mg of DNAse1, and two tablets of Roche protease inhibitor cocktail were added into the cell suspension.

Article Title: Modified Cav1.4 Expression in the Cacna1fnob2 Mouse Due to Alternative Splicing of an ETn Inserted in Exon 2
Article Snippet: Each fragment was ligated into the T-easy vector system (Promega), the sequence was confirmed, and the fragment was subcloned into the expression vector pCDNA3.1neo. .. After a two-day incubation the cells were lysed on ice with lysis buffer (300 mM NaCl, 50 mM Tris pH 7.5, 0.1% Triton X-100) containing a protease-inhibitor cocktail (complete Mini EDTA-free, Roche).

Software:

Article Title: Reduced insulin action in muscle of high fat diet rats over the diurnal cycle is not associated with defective insulin signaling
Article Snippet: 2.8 Immunoblotting Frozen, powdered tissue was homogenized by probe homogenizer in RIPA buffer containing protease inhibitor cocktail (1 × EDTA free; Roche) and phosphatase inhibitors (10 mM NaF, 1 mM Na3 VO4 ). .. Densitometry analysis was performed using ImageJ software (NIH; http://imagej.nih.gov/ij/ ).

Positron Emission Tomography:

Article Title: 3.5? cryoEM Structure of Hepatitis B Virus Core Assembled from Full-Length Core Protein
Article Snippet: The amplified gene was double-digested with Nde I and Bam HI, and subsequently cloned into the expression vector pET-11a from Novagen. .. 60mg of chicken lysozyme, 15mg of DNAse1, and two tablets of Roche protease inhibitor cocktail were added into the cell suspension.

Homogenization:

Article Title: Distinct perinatal features of the hyperpolarization-activated non-selective cation current Ih in the rat cortical plate
Article Snippet: .. Tissue homogenization was performed in lysis buffer containing 20 mM Tris–HCl (pH 7.5), 0.25 M sucrose, 1 mM EGTA, and 5 mM EDTA which was supplemented with a protease inhibitor cocktail (PIC Complete, diluted according to the manufacturer; Roche Applied Science, Mannheim, Germany) and a phosphatase inhibitor cocktail (PhosSTOP, diluted according to the manufacturer; Roche). .. As positive controls we used protein extracts from HEK293 cells over-expressing either pIRES2-dsRed-rHCN1 [ ], pIRES2-eGFP-rHCN2 (kindly provided by Shigetada Nakanishi), rHCN3-pcDNA1 (kindly provided by Prof. Müller, Research Centre Jülich), or hHCN4-pcDNA3 (kindly provided by Juliane Stieber, University of Erlangen).

Concentration Assay:

Article Title: Ezrin is a Major Regulator of Membrane Tension in Epithelial Cells
Article Snippet: After washing cells three times with PBS− , cell lysis was achieved by incubation with radioimmunoprecipitation (RIPA) buffer containing a protease inhibitor cocktail (cOmplete EDTA-free tablets; Roche Diagnostics, Mannheim, Germany) for 5 min at 0 °C. .. The primary antibody (ezrin mouse IgG1; BD Biosciences, Heidelberg, Germany) was diluted with TBT buffer containing 5% (w /v ) lowfat dry milk to a concentration of 500 ng/mL and incubated with the nitrocellulose membrane overnight at 4 °C.

Article Title: 3.5? cryoEM Structure of Hepatitis B Virus Core Assembled from Full-Length Core Protein
Article Snippet: After IPTG was added into the culture medium with a final concentration of 0.2mM when A600 of medium reached 0.8, the bacterial cells were grown for another 4 hours to induce HBV core protein production. .. 60mg of chicken lysozyme, 15mg of DNAse1, and two tablets of Roche protease inhibitor cocktail were added into the cell suspension.

Staining:

Article Title: Oesophagostomum dentatum Extract Modulates T Cell-Dependent Immune Responses to Bystander Antigens and Prevents the Development of Allergy in Mice
Article Snippet: Bronchoalvelar Lavage and Differential Cell Counts Bronchoalveolar lavage fluid (BALF) was collected by cannulating the trachea and injecting/recovering 2×1 ml PBS containing a protease inhibitor cocktail (Complete® Protease Inhibitor cocktail Tablets; Roche, Mannheim, Germany). .. The cell pellet was resuspended in 200 µl of PBS; total leukocytes were counted and cytospins (4×104 cells; Shandon Cytospin®, Shandon Southern Instruments, USA) were stained with haematoxylin and eosin (H & E Hemacolor®, Merck, Darmstadt, Germany) for differential cell counts (200 cells were counted per cytospin).

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  • 96
    Roche immunoprecipitation buffer
    CNTNAP2 interacts with CASK at the plasma membrane in cortical GABAergic interneurons (a) Only yeast cells co-expressing CNTNAP2 bait and CASK prey constructs grow on high stringency yeast plates (QDO/X/A). (b–c) Cropped western blots of <t>co-immunoprecipitation</t> experiments with CASK and CNTNAP2 in mouse cortex. (d) Cropped western blots showing co-immunoprecipitation of various FLAG-CNTNAP2 truncation mutants ( Supplementary Figure 5a ; red lines) with untagged, full-length CASK in HEK293T cells. (e) Representative cropped western blots of membrane/cytosol fractions of HEK293T cells expressing pCS2-FLAG + CASK-mCherry (CASK), FLAG-CNTNAP2 + CASK-mCherry (CNTNAP2 + CASK), or FLAG-CNTNAP2 + CASKΔPDZ-mCherry (CNTNAP2 + CASKΔPDZ) and subsequent quantification of protein localization (CASK vs. CNTNAP2 + CASK vs. CNTNAP2 + CASKΔPDZ: 6 independent experiments; CASK alone vs. CASKΔPDZ alone: 3 independent experiments). Percentages were calculated by dividing the densitometry value of CASK/CNTNAP2 in either membrane or cytosol fraction by the summation of both. (f) Representative SIM image of endogenous CNTNAP2 and CASK co-localization (white) on a GFP-transfected interneuronal dendrite (scale bar = 5 μm). (g) Histogram showing distribution of CASK/CNTNAP2 co-localized puncta relative to the dendrite’s lateral edge from (f) (CASK colocalized with CNTNAP2: n = 79 puncta from 3 cultures; CNTNAP2 colocalized with CASK: n = 70 puncta from 3 cultures). (h) Representative confocal image showing PLA signal from endogenous CASK/CNTNAP2 staining, which occurs only when CASK and CNTNAP2 primary antibodies are both applied (scale bar = 1 μm). (i) Cropped immunoblots of subcellular fractionations from adult mouse forebrain probed with CNTNAP2, CASK, and β-tubulin. CNTNAP2 and CASK, but not β-tubulin, are found in the washed membrane fraction (S5; red box). (j) Cropped western blot of time course and (k) quantification of CASK expression in cultured cortical neurons (n = 3 independent experiments). Values are means ± SEM. * P≤0.05, ** P≤0.01, *** P≤0.001; one-way ANOVA with Bonferroni’s correction (k, top graph; e). Student’s t-test (middle and bottom graphs; e).
    Immunoprecipitation Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 96/100, based on 228 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Roche complete mini protease inhibitor cocktail
    Expression of conventional breast cancer biomarkers and selected multidrug resistance genes. Cell lysates were prepared in radioimmunoprecipitation assay buffer supplemented with <t>cOmplete</t> Mini protease inhibitor and PhosSTOP phosphatase inhibitor. Quantities (10–50 μg) of total protein were run on a 10 % gel (MDR1), 4–20 % precast gels (epidermal growth factor receptor [EGFR], oestrogen receptor [ER], progesterone receptor [PR], type II topoisomerase [TOPO IIα]) and Any kD Mini-PROTEAN TGX Precast Protein Gels (human epidermal growth factor receptor 2 [HER2], HER3), transferred onto polyvinylidene fluoride membrane and developed using chemiluminescence substrate. Nat native, Epi-R epirubicin-resistant, GAPDH glyceraldehyde-3-phosphate dehydrogenase
    Complete Mini Protease Inhibitor Cocktail, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 350 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/complete mini protease inhibitor cocktail/product/Roche
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    Price from $9.99 to $1999.99
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    94
    Roche pbs containing protease inhibitor cocktail
    ST398 CA-MRSA virulence in a mouse model. The seven ST398 CA-MRSA isolates were compared with closely related ST398 MSSA, all ST398 LA-MSSA obtained for this study, and representative Chinese ST5 and ST239 HA-MRSA strains by in vivo analysis of virulence. In addition, two USA300 CA-MRSA strains (SF8300, LAC) and the genome-sequenced LA-MRSA S0385 standard strain were used in the comparisons. Isolate selection for the MSSA and HA-MRSA groups was performed, as in a previous study [ 26 ], by selecting isolates whose average PSM production was similar to that of the entire group and thus reflects the average virulence potential. Notably, for the in vivo infection studies, the ST398 CA-MRSA, MSSA, and ST5/ST239 HA-MRSA isolates were obtained from the corresponding type of human infection (respiratory and skin infection, respectively). See Additional file 1 : Table S2 for isolates that were compared and their characteristics. a Mouse skin infection model. Mice received 10 7 live S. aureus or <t>PBS</t> alone in the right flank by subcutaneous injection. Skin abscesses (length × width) were measured 48 h after infection. b Mouse lung infection model. We pipetted 4 × 10 9 CFU/40 μl S. aureus into the nares of the anesthetized mice. All mice were euthanized 48 h after inoculation and CFU in the lung tissue were determined. a , b Three mice were infected with each strain; the shown symbols represent the average value obtained from three mice. c – f Inflammatory <t>cytokine</t> expression in ST398 CA-MRSA isolates in comparison. The inflammatory cytokines TNF-α and IL-6 were determined in skin ( c , d ) or lung tissue ( e , f ). a – f Statistical analysis is by one-way ANOVA with Dunnett’s post test versus the ST398 CA-MRSA group. * p
    Pbs Containing Protease Inhibitor Cocktail, supplied by Roche, used in various techniques. Bioz Stars score: 94/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    CNTNAP2 interacts with CASK at the plasma membrane in cortical GABAergic interneurons (a) Only yeast cells co-expressing CNTNAP2 bait and CASK prey constructs grow on high stringency yeast plates (QDO/X/A). (b–c) Cropped western blots of co-immunoprecipitation experiments with CASK and CNTNAP2 in mouse cortex. (d) Cropped western blots showing co-immunoprecipitation of various FLAG-CNTNAP2 truncation mutants ( Supplementary Figure 5a ; red lines) with untagged, full-length CASK in HEK293T cells. (e) Representative cropped western blots of membrane/cytosol fractions of HEK293T cells expressing pCS2-FLAG + CASK-mCherry (CASK), FLAG-CNTNAP2 + CASK-mCherry (CNTNAP2 + CASK), or FLAG-CNTNAP2 + CASKΔPDZ-mCherry (CNTNAP2 + CASKΔPDZ) and subsequent quantification of protein localization (CASK vs. CNTNAP2 + CASK vs. CNTNAP2 + CASKΔPDZ: 6 independent experiments; CASK alone vs. CASKΔPDZ alone: 3 independent experiments). Percentages were calculated by dividing the densitometry value of CASK/CNTNAP2 in either membrane or cytosol fraction by the summation of both. (f) Representative SIM image of endogenous CNTNAP2 and CASK co-localization (white) on a GFP-transfected interneuronal dendrite (scale bar = 5 μm). (g) Histogram showing distribution of CASK/CNTNAP2 co-localized puncta relative to the dendrite’s lateral edge from (f) (CASK colocalized with CNTNAP2: n = 79 puncta from 3 cultures; CNTNAP2 colocalized with CASK: n = 70 puncta from 3 cultures). (h) Representative confocal image showing PLA signal from endogenous CASK/CNTNAP2 staining, which occurs only when CASK and CNTNAP2 primary antibodies are both applied (scale bar = 1 μm). (i) Cropped immunoblots of subcellular fractionations from adult mouse forebrain probed with CNTNAP2, CASK, and β-tubulin. CNTNAP2 and CASK, but not β-tubulin, are found in the washed membrane fraction (S5; red box). (j) Cropped western blot of time course and (k) quantification of CASK expression in cultured cortical neurons (n = 3 independent experiments). Values are means ± SEM. * P≤0.05, ** P≤0.01, *** P≤0.001; one-way ANOVA with Bonferroni’s correction (k, top graph; e). Student’s t-test (middle and bottom graphs; e).

    Journal: Molecular psychiatry

    Article Title: CNTNAP2 stabilizes interneuron dendritic arbors through CASK

    doi: 10.1038/s41380-018-0027-3

    Figure Lengend Snippet: CNTNAP2 interacts with CASK at the plasma membrane in cortical GABAergic interneurons (a) Only yeast cells co-expressing CNTNAP2 bait and CASK prey constructs grow on high stringency yeast plates (QDO/X/A). (b–c) Cropped western blots of co-immunoprecipitation experiments with CASK and CNTNAP2 in mouse cortex. (d) Cropped western blots showing co-immunoprecipitation of various FLAG-CNTNAP2 truncation mutants ( Supplementary Figure 5a ; red lines) with untagged, full-length CASK in HEK293T cells. (e) Representative cropped western blots of membrane/cytosol fractions of HEK293T cells expressing pCS2-FLAG + CASK-mCherry (CASK), FLAG-CNTNAP2 + CASK-mCherry (CNTNAP2 + CASK), or FLAG-CNTNAP2 + CASKΔPDZ-mCherry (CNTNAP2 + CASKΔPDZ) and subsequent quantification of protein localization (CASK vs. CNTNAP2 + CASK vs. CNTNAP2 + CASKΔPDZ: 6 independent experiments; CASK alone vs. CASKΔPDZ alone: 3 independent experiments). Percentages were calculated by dividing the densitometry value of CASK/CNTNAP2 in either membrane or cytosol fraction by the summation of both. (f) Representative SIM image of endogenous CNTNAP2 and CASK co-localization (white) on a GFP-transfected interneuronal dendrite (scale bar = 5 μm). (g) Histogram showing distribution of CASK/CNTNAP2 co-localized puncta relative to the dendrite’s lateral edge from (f) (CASK colocalized with CNTNAP2: n = 79 puncta from 3 cultures; CNTNAP2 colocalized with CASK: n = 70 puncta from 3 cultures). (h) Representative confocal image showing PLA signal from endogenous CASK/CNTNAP2 staining, which occurs only when CASK and CNTNAP2 primary antibodies are both applied (scale bar = 1 μm). (i) Cropped immunoblots of subcellular fractionations from adult mouse forebrain probed with CNTNAP2, CASK, and β-tubulin. CNTNAP2 and CASK, but not β-tubulin, are found in the washed membrane fraction (S5; red box). (j) Cropped western blot of time course and (k) quantification of CASK expression in cultured cortical neurons (n = 3 independent experiments). Values are means ± SEM. * P≤0.05, ** P≤0.01, *** P≤0.001; one-way ANOVA with Bonferroni’s correction (k, top graph; e). Student’s t-test (middle and bottom graphs; e).

    Article Snippet: Expression Time Course Cortices from CD1 WT mice aged P0, P14, P28, 4 months, and 6 months (sex not determined at P0, P14; males for P28, 4 months, 6 months) were homogenized in immunoprecipitation buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.5% Triton X-100 with Roche protease inhibitor cocktail) and solubilized for 1 hour at 4°C.

    Techniques: Expressing, Construct, Western Blot, Immunoprecipitation, Transfection, Proximity Ligation Assay, Staining, Cell Culture

    Hedgehog signaling leads to reduced nuclear Sufu protein levels and Sufu dissociation from Gli2 and Gli3 primarily in the nucleus. ( A ) Western blot analysis and quantification of Sufu protein levels in the nuclear and cytoplasmic fractions derived from MEFs treated with Shh-conditioned medium. Cytoplasmic tubulin and nuclear lamin A were used to assess the purity of cytoplasmic and nuclear fractions. Nuclear Sufu protein levels were reduced by 60% upon Hh pathway activation, while cytoplasmic Sufu levels were largely unaltered. ( B ) Western blot analysis and quantification of Sufu protein levels in the nuclear and cytoplasmic fractions in the presence of Shh-conditioned medium and proteasome inhibitor MG132. Nuclear Sufu protein levels were notably restored upon MG132 addition. ( C ) Western blot analysis and quantification of immunoprecipitated Sufu, Gli2, and Gli3 using lysates from the nuclear and cytoplasmic fractions derived from MEFs expressing Flag-tagged Sufu. The amount of coimmunoprecipitated Gli2 and Gli3 by Sufu was significantly reduced in the nuclear fraction but only marginally decreased in the cytoplasmic fraction at indicated time points after Hh stimulation. (In) Input; (IP) immunoprecipitation. (*) P

    Journal: Genes & Development

    Article Title: Regulation of Sufu activity by p66β and Mycbp provides new insight into vertebrate Hedgehog signaling

    doi: 10.1101/gad.249425.114

    Figure Lengend Snippet: Hedgehog signaling leads to reduced nuclear Sufu protein levels and Sufu dissociation from Gli2 and Gli3 primarily in the nucleus. ( A ) Western blot analysis and quantification of Sufu protein levels in the nuclear and cytoplasmic fractions derived from MEFs treated with Shh-conditioned medium. Cytoplasmic tubulin and nuclear lamin A were used to assess the purity of cytoplasmic and nuclear fractions. Nuclear Sufu protein levels were reduced by 60% upon Hh pathway activation, while cytoplasmic Sufu levels were largely unaltered. ( B ) Western blot analysis and quantification of Sufu protein levels in the nuclear and cytoplasmic fractions in the presence of Shh-conditioned medium and proteasome inhibitor MG132. Nuclear Sufu protein levels were notably restored upon MG132 addition. ( C ) Western blot analysis and quantification of immunoprecipitated Sufu, Gli2, and Gli3 using lysates from the nuclear and cytoplasmic fractions derived from MEFs expressing Flag-tagged Sufu. The amount of coimmunoprecipitated Gli2 and Gli3 by Sufu was significantly reduced in the nuclear fraction but only marginally decreased in the cytoplasmic fraction at indicated time points after Hh stimulation. (In) Input; (IP) immunoprecipitation. (*) P

    Article Snippet: Cells were collected at 48 h post-transfection and lysed in immunoprecipitation buffer (1% Triton X-100, 150 mM NaCl, 50mM Tris-Cl at pH 7.5, 1 mM EDTA, protease inhibitor cocktail [Roche], PhosSTOP [Roche]).

    Techniques: Western Blot, Derivative Assay, Activation Assay, Immunoprecipitation, Expressing

    A proteomic approach to identify Sufu-interacting proteins uncovers p66β and Mycbp. ( A ) Coomassie Blue-stained gel of control and Sufu immunoprecipitates treated with mock- or Shh-conditioned medium. Distinct bands were detected and were candidates for new Sufu-interacting proteins. Numbers at the right indicate locations of protein size standards. Large-scale immunoprecipitation (IP) and mass spectrometry were performed to identify new Sufu-interacting proteins and Sufu phosphorylation sites. Mass spectrometric analysis was performed directly on immunoprecipitates or specific bands cut out from SDS-PAGE gels. Immunoprecipitation and mass spectrometric analysis were repeated multiple times to eliminate nonspecific Sufu-binding proteins. ( B ) Western blot analysis of Sufu immunoprecipitates probed with anti-Sufu, anti-Gli2, and Gli3 antibodies. Endogenous Gli2 and Gli3 were detected in Sufu immunoprecipitates (but not in the control), suggesting that a physiologically relevant protein complex was pulled down. ( C ). ( D ) Western blot analysis of proteins pulled down by Sufu from lysates expressing Sufu and the indicated proteins, which were epitope-tagged. Both p66β and Mycbp physically interacted with Sufu by coimmunoprecipitation. Fu and Prc1 served as negative controls. ( E , top panels) Western blot analysis of Sufu immunoprecipitates using lysates derived from MEFs expressing Flag-tagged Sufu. Endogenous p66β and HDAC1 were coimmunoprecipitated. In contrast, HDAC2 and RBBP7/4 could not be detected in Sufu immunoprecipitates. ( Bottom panels) Western blot analysis of endogenous Sufu immunoprecipitated by an anti-Sufu antibody. p66β was coimmunoprecipitated by Sufu in wild-type MEFs but not in Sufu -deficient MEFs. p66β/Sufu interaction was not altered by Hh stimulation (Supplemental Fig. S6). ( F–H ) Immunofluorescence studies to assess the subcellular distribution of p66β and Mycbp. MEFs were transfected or transduced with p66β- and Mycbp-expressing constructs. p66β and Mycbp localized to the nucleus (marked by DAPI) of Hh-responsive cells. Cytoplasmic expression of Mycbp was also detected. Acetylated (Ac)-tubulin marks the primary cilium. Interestingly, Mycbp immunoreactivity can also be detected at the base of the cilium (white arrow).

    Journal: Genes & Development

    Article Title: Regulation of Sufu activity by p66β and Mycbp provides new insight into vertebrate Hedgehog signaling

    doi: 10.1101/gad.249425.114

    Figure Lengend Snippet: A proteomic approach to identify Sufu-interacting proteins uncovers p66β and Mycbp. ( A ) Coomassie Blue-stained gel of control and Sufu immunoprecipitates treated with mock- or Shh-conditioned medium. Distinct bands were detected and were candidates for new Sufu-interacting proteins. Numbers at the right indicate locations of protein size standards. Large-scale immunoprecipitation (IP) and mass spectrometry were performed to identify new Sufu-interacting proteins and Sufu phosphorylation sites. Mass spectrometric analysis was performed directly on immunoprecipitates or specific bands cut out from SDS-PAGE gels. Immunoprecipitation and mass spectrometric analysis were repeated multiple times to eliminate nonspecific Sufu-binding proteins. ( B ) Western blot analysis of Sufu immunoprecipitates probed with anti-Sufu, anti-Gli2, and Gli3 antibodies. Endogenous Gli2 and Gli3 were detected in Sufu immunoprecipitates (but not in the control), suggesting that a physiologically relevant protein complex was pulled down. ( C ). ( D ) Western blot analysis of proteins pulled down by Sufu from lysates expressing Sufu and the indicated proteins, which were epitope-tagged. Both p66β and Mycbp physically interacted with Sufu by coimmunoprecipitation. Fu and Prc1 served as negative controls. ( E , top panels) Western blot analysis of Sufu immunoprecipitates using lysates derived from MEFs expressing Flag-tagged Sufu. Endogenous p66β and HDAC1 were coimmunoprecipitated. In contrast, HDAC2 and RBBP7/4 could not be detected in Sufu immunoprecipitates. ( Bottom panels) Western blot analysis of endogenous Sufu immunoprecipitated by an anti-Sufu antibody. p66β was coimmunoprecipitated by Sufu in wild-type MEFs but not in Sufu -deficient MEFs. p66β/Sufu interaction was not altered by Hh stimulation (Supplemental Fig. S6). ( F–H ) Immunofluorescence studies to assess the subcellular distribution of p66β and Mycbp. MEFs were transfected or transduced with p66β- and Mycbp-expressing constructs. p66β and Mycbp localized to the nucleus (marked by DAPI) of Hh-responsive cells. Cytoplasmic expression of Mycbp was also detected. Acetylated (Ac)-tubulin marks the primary cilium. Interestingly, Mycbp immunoreactivity can also be detected at the base of the cilium (white arrow).

    Article Snippet: Cells were collected at 48 h post-transfection and lysed in immunoprecipitation buffer (1% Triton X-100, 150 mM NaCl, 50mM Tris-Cl at pH 7.5, 1 mM EDTA, protease inhibitor cocktail [Roche], PhosSTOP [Roche]).

    Techniques: Staining, Immunoprecipitation, Mass Spectrometry, SDS Page, Binding Assay, Western Blot, Expressing, Derivative Assay, Immunofluorescence, Transfection, Transduction, Construct

    Expression of conventional breast cancer biomarkers and selected multidrug resistance genes. Cell lysates were prepared in radioimmunoprecipitation assay buffer supplemented with cOmplete Mini protease inhibitor and PhosSTOP phosphatase inhibitor. Quantities (10–50 μg) of total protein were run on a 10 % gel (MDR1), 4–20 % precast gels (epidermal growth factor receptor [EGFR], oestrogen receptor [ER], progesterone receptor [PR], type II topoisomerase [TOPO IIα]) and Any kD Mini-PROTEAN TGX Precast Protein Gels (human epidermal growth factor receptor 2 [HER2], HER3), transferred onto polyvinylidene fluoride membrane and developed using chemiluminescence substrate. Nat native, Epi-R epirubicin-resistant, GAPDH glyceraldehyde-3-phosphate dehydrogenase

    Journal: Breast Cancer Research : BCR

    Article Title: Downregulation of histone H2A and H2B pathways is associated with anthracycline sensitivity in breast cancer

    doi: 10.1186/s13058-016-0676-6

    Figure Lengend Snippet: Expression of conventional breast cancer biomarkers and selected multidrug resistance genes. Cell lysates were prepared in radioimmunoprecipitation assay buffer supplemented with cOmplete Mini protease inhibitor and PhosSTOP phosphatase inhibitor. Quantities (10–50 μg) of total protein were run on a 10 % gel (MDR1), 4–20 % precast gels (epidermal growth factor receptor [EGFR], oestrogen receptor [ER], progesterone receptor [PR], type II topoisomerase [TOPO IIα]) and Any kD Mini-PROTEAN TGX Precast Protein Gels (human epidermal growth factor receptor 2 [HER2], HER3), transferred onto polyvinylidene fluoride membrane and developed using chemiluminescence substrate. Nat native, Epi-R epirubicin-resistant, GAPDH glyceraldehyde-3-phosphate dehydrogenase

    Article Snippet: WCL were prepared by adding equal volumes of 2× RIPA buffer supplemented with 25 U of Benzonase Nuclease (Sigma-Aldrich) and cOmplete Mini Protease Inhibitor Cocktail (Roche), incubating on ice for 30 minutes and sonicating for 15 minutes with 30-second on-off intervals.

    Techniques: Expressing, Radio Immunoprecipitation, Protease Inhibitor

    ST398 CA-MRSA virulence in a mouse model. The seven ST398 CA-MRSA isolates were compared with closely related ST398 MSSA, all ST398 LA-MSSA obtained for this study, and representative Chinese ST5 and ST239 HA-MRSA strains by in vivo analysis of virulence. In addition, two USA300 CA-MRSA strains (SF8300, LAC) and the genome-sequenced LA-MRSA S0385 standard strain were used in the comparisons. Isolate selection for the MSSA and HA-MRSA groups was performed, as in a previous study [ 26 ], by selecting isolates whose average PSM production was similar to that of the entire group and thus reflects the average virulence potential. Notably, for the in vivo infection studies, the ST398 CA-MRSA, MSSA, and ST5/ST239 HA-MRSA isolates were obtained from the corresponding type of human infection (respiratory and skin infection, respectively). See Additional file 1 : Table S2 for isolates that were compared and their characteristics. a Mouse skin infection model. Mice received 10 7 live S. aureus or PBS alone in the right flank by subcutaneous injection. Skin abscesses (length × width) were measured 48 h after infection. b Mouse lung infection model. We pipetted 4 × 10 9 CFU/40 μl S. aureus into the nares of the anesthetized mice. All mice were euthanized 48 h after inoculation and CFU in the lung tissue were determined. a , b Three mice were infected with each strain; the shown symbols represent the average value obtained from three mice. c – f Inflammatory cytokine expression in ST398 CA-MRSA isolates in comparison. The inflammatory cytokines TNF-α and IL-6 were determined in skin ( c , d ) or lung tissue ( e , f ). a – f Statistical analysis is by one-way ANOVA with Dunnett’s post test versus the ST398 CA-MRSA group. * p

    Journal: Genome Medicine

    Article Title: Detection and analysis of methicillin-resistant human-adapted sequence type 398 allows insight into community-associated methicillin-resistant Staphylococcus aureus evolution

    doi: 10.1186/s13073-018-0514-9

    Figure Lengend Snippet: ST398 CA-MRSA virulence in a mouse model. The seven ST398 CA-MRSA isolates were compared with closely related ST398 MSSA, all ST398 LA-MSSA obtained for this study, and representative Chinese ST5 and ST239 HA-MRSA strains by in vivo analysis of virulence. In addition, two USA300 CA-MRSA strains (SF8300, LAC) and the genome-sequenced LA-MRSA S0385 standard strain were used in the comparisons. Isolate selection for the MSSA and HA-MRSA groups was performed, as in a previous study [ 26 ], by selecting isolates whose average PSM production was similar to that of the entire group and thus reflects the average virulence potential. Notably, for the in vivo infection studies, the ST398 CA-MRSA, MSSA, and ST5/ST239 HA-MRSA isolates were obtained from the corresponding type of human infection (respiratory and skin infection, respectively). See Additional file 1 : Table S2 for isolates that were compared and their characteristics. a Mouse skin infection model. Mice received 10 7 live S. aureus or PBS alone in the right flank by subcutaneous injection. Skin abscesses (length × width) were measured 48 h after infection. b Mouse lung infection model. We pipetted 4 × 10 9 CFU/40 μl S. aureus into the nares of the anesthetized mice. All mice were euthanized 48 h after inoculation and CFU in the lung tissue were determined. a , b Three mice were infected with each strain; the shown symbols represent the average value obtained from three mice. c – f Inflammatory cytokine expression in ST398 CA-MRSA isolates in comparison. The inflammatory cytokines TNF-α and IL-6 were determined in skin ( c , d ) or lung tissue ( e , f ). a – f Statistical analysis is by one-way ANOVA with Dunnett’s post test versus the ST398 CA-MRSA group. * p

    Article Snippet: Length (L) and width (W) values were used to calculate the area of lesions with the formula L × W. Mouse skin tissue of the same size was taken from the abscess and PBS control groups and homogenated with glass beads with PBS-containing protease inhibitor cocktail (Roche) for cytokine detection.

    Techniques: In Vivo, Selection, Infection, Mouse Assay, Injection, Expressing