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Roche cacl2
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Centrifugation:

Article Title: A switch in surface polymer biogenesis triggers growth-phase-dependent and antibiotic-induced bacteriolysis
Article Snippet: Complete protoplasting was monitored by light microscopy. .. Protoplasts were pelleted by centrifugation at 5000 g for 5 min and resuspended in 2 ml cold hypotonic buffer to lyse them (20 mM HEPES (Na+ ), pH 8.0, 100 mM NaCl, 1 mM dithiothreitol (DTT), 1 mM MgCl2 , 1 mM CaCl2 , 2X complete protease inhibitors (Roche), 6 μg/ml RNAse A, 6 μg/ml DNAse). .. Unbroken spheroplasts were removed by centrifugation 5,000 rpm for 10 min, and then the membrane fraction was collected by ultracentrifugation at 100,000 g for 1 hr at 4°C.

Article Title: Alzheimer Amyloid-? Oligomer Bound to Post-Synaptic Prion Protein Activates Fyn to Impair Neurons
Article Snippet: Rat forebrains were homogenized in ice-cold 5 mM NaHEPES, pH7.4, 1 mM MgCl2 , 0.5 mM CaCl2 , complete protease inhibitor cocktail (Roche), phosphatase inhibitor (Roche) with a glass/Teflon pestel. .. P2 was resuspended in Buffer B (0.32 M sucrose, 6 mM TrisHCl, pH 8.0, complete protease inhibitor cocktail (Roche), phosphatase inhibitor (Roche)).

Article Title: Spermine synthase deficiency causes lysosomal dysfunction and oxidative stress in models of Snyder-Robinson syndrome
Article Snippet: Specifically, histogram plots in Fig. and Supplementary Fig. are analyzed using ImageJ Plot Profile and surface plots in Fig. and Supplementary Fig. are plotted using ImageJ Interactive 3D Surface Plot plugin. .. For immunoblot analysis of cathepsins, tissues were homogenized on ice in lysis buffer containing 20 mM HEPES, 100 mM NaCl, 1 mM CaCl2 , 0.5% Triton X-100 and cOmplete protease inhibitor cocktail (Roche), followed by 10 min centrifugation at 10,000×g at 4 °C. .. The supernatants were then mixed with Laemmli sample buffer and heated at 95 °C for 10 min. Proteins were separated on a Bis-Tris 10% gel and transferred to a nitrocellulose membrane.

Article Title: Elevated Expression of Osteopontin May Be Related to Adipose Tissue Macrophage Accumulation and Liver Steatosis in Morbid Obesity
Article Snippet: Mouse livers were perfused first with a HEPES buffer containing 8 g/l NaCl, 33 mg/l Na2 HPO4 , 200 mg/l KCl, and 2.38 g/l HEPES, pH 7.65, for 2 min at 5 ml/min and then with HEPES buffer supplemented with 1.5 g/l CaCl2 and 25 mg/l liberase (Roche Diagnostics) for 7 min at 5 ml/min. .. Mouse livers were perfused first with a HEPES buffer containing 8 g/l NaCl, 33 mg/l Na2 HPO4 , 200 mg/l KCl, and 2.38 g/l HEPES, pH 7.65, for 2 min at 5 ml/min and then with HEPES buffer supplemented with 1.5 g/l CaCl2 and 25 mg/l liberase (Roche Diagnostics) for 7 min at 5 ml/min.

Article Title: Regulation of HLTF-mediated PCNA polyubiquitination by RFC and PCNA monoubiquitination levels determines choice of damage tolerance pathway
Article Snippet: The cells were suspended in lysis buffer [20 mM Tris–HCl (pH 7.6), 150 mM KCl, 10% glycerol, 0.5% NP-40, 1.5 mM MgCl2 , 0.2 μg/ml antipain, 0.2 μg/ml aprotinin, 0.1 μg/ml leupeptin, 0.08 μg/ml pepstatin, 0.05 mM EGTA and 0.25 mM phenylmethylsulfonyl fluoride (PMSF)], and soluble materials were separated by centrifugation. .. The precipitates (insoluble fraction) were suspended in micrococcal nuclease buffer [20 mM Tris–HCl (pH 7.6), 100 mM KCl, 0.3 M sucrose, 0.1% Triton X-100, 2 mM MgCl2 , 1 mM CaCl2 and cOmplete™ EDTA-free Protease Inhibitor Cocktail (Roche)] and digested with MNase at 25°C for 10 min.

Stable Transfection:

Article Title: Regulation of HLTF-mediated PCNA polyubiquitination by RFC and PCNA monoubiquitination levels determines choice of damage tolerance pathway
Article Snippet: Then, stable cell lines expressing 3× FLAG-tagged HLTF (HLTF −/− /3xFLAG HLTF ) and a vector-transfected cell line (HLTF −/− /vector) were established. .. The precipitates (insoluble fraction) were suspended in micrococcal nuclease buffer [20 mM Tris–HCl (pH 7.6), 100 mM KCl, 0.3 M sucrose, 0.1% Triton X-100, 2 mM MgCl2 , 1 mM CaCl2 and cOmplete™ EDTA-free Protease Inhibitor Cocktail (Roche)] and digested with MNase at 25°C for 10 min.

Cytometry:

Article Title: Selective expansion of myeloid and NK cells in humanized mice yields human-like vaccine responses
Article Snippet: Fetal liver was homogenized and incubated in digestion medium (HBSS) with 0.1% collagenase IV (Sigma-Aldrich, Darmstadt, Germany), 40 mM HEPES, 2 M CaCl2 , and 2 U/ml DNAse I (Roche, Basel, Switzerland) for 30 min at 37 °C. .. Human CD34+ HSC were isolated using a CD34+ HSC isolation kit (Stem Cell Technologies, Vancouver, British Columbia, Canada) according to the manufacturer’ protocol.

Article Title: Single-cell tracking of flavivirus RNA uncovers species-specific interactions with the immune system dictating disease outcome
Article Snippet: Fetal liver was homogenized and incubated in digestion medium (HBSS with 0.1% collagenase IV (Sigma), 40 mM HEPES, 2 M CaCl2 and 2 U ml−1 DNAse I (Roche) for 30 min at 37 °C. .. Human CD34+ HSC were isolated using a CD34+ HSC isolation kit (Stem Cell Technologies) according to the manufacturer' protocol.

Blocking Assay:

Article Title: The Immune Interplay between Thyroid Papillary Carcinoma and Hepatic Fibrosis
Article Snippet: Whole liver protein extracts were prepared in liver homogenization buffer (50 mmol/L Tris–HCl [pH 7.6], 0.25% Triton-X 100, 0.15 M NaCl, 10 mM CaCl2 and complete mini EDTA-free protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany)). .. Whole liver protein extracts were prepared in liver homogenization buffer (50 mmol/L Tris–HCl [pH 7.6], 0.25% Triton-X 100, 0.15 M NaCl, 10 mM CaCl2 and complete mini EDTA-free protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany)).

Article Title: EGF receptor kinase suppresses ciliogenesis through activation of USP8 deubiquitinase
Article Snippet: The embryo or fishes samples were soaked in a cacodylate-buffered solution containing 2 mM CaCl2 , 30% sucrose, and protease inhibitor cocktail tablets (Roche Diagnostics, Mannheim, Germany) at 4 ℃ for overnight. .. Then, the samples were quickly frozen in OCT compound (Sakura Finetek, Tokyo, Japan) and 12-μm thickness serial cryostat sections were cut and mounted on glass slides, then quickly blocked by 0.1 M phosphate buffer (pH 7.4) containing 4% Block Ace (DS Pharma Biomedical), protease cocktail, and 0.02% saponin, and incubated at room temperature (RT) for 20 min. Then, the samples were incubated at 4 ℃ for 1 overnight for primary Abs, and the 2 h at RT for secondary Abs.

Article Title: Spermine synthase deficiency causes lysosomal dysfunction and oxidative stress in models of Snyder-Robinson syndrome
Article Snippet: For immunoblot analysis of cathepsins, tissues were homogenized on ice in lysis buffer containing 20 mM HEPES, 100 mM NaCl, 1 mM CaCl2 , 0.5% Triton X-100 and cOmplete protease inhibitor cocktail (Roche), followed by 10 min centrifugation at 10,000×g at 4 °C. .. For immunoblot analysis of cathepsins, tissues were homogenized on ice in lysis buffer containing 20 mM HEPES, 100 mM NaCl, 1 mM CaCl2 , 0.5% Triton X-100 and cOmplete protease inhibitor cocktail (Roche), followed by 10 min centrifugation at 10,000×g at 4 °C.

Incubation:

Article Title: Early preclinical detection of prions in the skin of prion-infected animals
Article Snippet: The samples were carefully cleaned in Tris-buffered saline (TBS) containing 10 mM Tris-HCl, 133 mM NaCl, pH 7.4 for three times before processing of each specimen in order to avoid blood contamination. .. For western blotting, or sPMCA assay, the skin samples were incubated in TBS containing 2 mM CaCl2 and 0.25% (10% w/v) collagenase A (Roche) in a shaker at 37 °C for 4 h. Beads Beater was used to make tissue homogenates and the samples were then centrifuged at 500×g for 5 min. .. The supernatant (S1) was transferred to a clean tube for future use and discard the pellet (P1).

Article Title: The Immune Interplay between Thyroid Papillary Carcinoma and Hepatic Fibrosis
Article Snippet: Whole liver protein extracts were prepared in liver homogenization buffer (50 mmol/L Tris–HCl [pH 7.6], 0.25% Triton-X 100, 0.15 M NaCl, 10 mM CaCl2 and complete mini EDTA-free protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany)). .. Whole liver protein extracts were prepared in liver homogenization buffer (50 mmol/L Tris–HCl [pH 7.6], 0.25% Triton-X 100, 0.15 M NaCl, 10 mM CaCl2 and complete mini EDTA-free protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany)).

Article Title: Selective expansion of myeloid and NK cells in humanized mice yields human-like vaccine responses
Article Snippet: Human fetal livers (16–22 weeks of gestational age) were procured from Advanced Bioscience Resources (ABR), Inc. (Alameda, CA). .. Fetal liver was homogenized and incubated in digestion medium (HBSS) with 0.1% collagenase IV (Sigma-Aldrich, Darmstadt, Germany), 40 mM HEPES, 2 M CaCl2 , and 2 U/ml DNAse I (Roche, Basel, Switzerland) for 30 min at 37 °C. .. Human CD34+ HSC were isolated using a CD34+ HSC isolation kit (Stem Cell Technologies, Vancouver, British Columbia, Canada) according to the manufacturer’ protocol.

Article Title: Neutron-encoded mass signatures for multi-plexed proteome quantification
Article Snippet: The cell pellets were resuspended in 8 M urea, 50 mM Tris pH 8.0, 5 mM CaCl2 , and protease inhibitors (Roche). .. Lysate protein concentration was measured by BCA (Pierce).

Article Title: A switch in surface polymer biogenesis triggers growth-phase-dependent and antibiotic-induced bacteriolysis
Article Snippet: Protoplasts were generated by addition of 20 mg/ml lysozyme and 100 units mutanolysin (Sigma) and incubated at 37°C for 30 min. .. Protoplasts were pelleted by centrifugation at 5000 g for 5 min and resuspended in 2 ml cold hypotonic buffer to lyse them (20 mM HEPES (Na+ ), pH 8.0, 100 mM NaCl, 1 mM dithiothreitol (DTT), 1 mM MgCl2 , 1 mM CaCl2 , 2X complete protease inhibitors (Roche), 6 μg/ml RNAse A, 6 μg/ml DNAse).

Article Title: Single-cell tracking of flavivirus RNA uncovers species-specific interactions with the immune system dictating disease outcome
Article Snippet: Human fetal livers (16–22 weeks of gestational age) were procured from Advanced Bioscience Resources (ABR), Inc. (Alameda, CA). .. Fetal liver was homogenized and incubated in digestion medium (HBSS with 0.1% collagenase IV (Sigma), 40 mM HEPES, 2 M CaCl2 and 2 U ml−1 DNAse I (Roche) for 30 min at 37 °C. .. Human CD34+ HSC were isolated using a CD34+ HSC isolation kit (Stem Cell Technologies) according to the manufacturer' protocol.

Article Title: Spermine synthase deficiency causes lysosomal dysfunction and oxidative stress in models of Snyder-Robinson syndrome
Article Snippet: For immunoblot analysis of cathepsins, tissues were homogenized on ice in lysis buffer containing 20 mM HEPES, 100 mM NaCl, 1 mM CaCl2 , 0.5% Triton X-100 and cOmplete protease inhibitor cocktail (Roche), followed by 10 min centrifugation at 10,000×g at 4 °C. .. For immunoblot analysis of cathepsins, tissues were homogenized on ice in lysis buffer containing 20 mM HEPES, 100 mM NaCl, 1 mM CaCl2 , 0.5% Triton X-100 and cOmplete protease inhibitor cocktail (Roche), followed by 10 min centrifugation at 10,000×g at 4 °C.

Article Title: Elevated Expression of Osteopontin May Be Related to Adipose Tissue Macrophage Accumulation and Liver Steatosis in Morbid Obesity
Article Snippet: Mouse livers were perfused first with a HEPES buffer containing 8 g/l NaCl, 33 mg/l Na2 HPO4 , 200 mg/l KCl, and 2.38 g/l HEPES, pH 7.65, for 2 min at 5 ml/min and then with HEPES buffer supplemented with 1.5 g/l CaCl2 and 25 mg/l liberase (Roche Diagnostics) for 7 min at 5 ml/min. .. Hepatocytes were then washed in NaCl 0.9%, isolated by centrifugation (5 min, 50g ), and frozen at −80°C before RNA extraction, as described below.

Article Title: Regulation of HLTF-mediated PCNA polyubiquitination by RFC and PCNA monoubiquitination levels determines choice of damage tolerance pathway
Article Snippet: After one wash with phosphate-buffered saline, cells were irradiated with UVC (15 J/m2 ), incubated under the above-described conditions for 3 h, and harvested. .. The precipitates (insoluble fraction) were suspended in micrococcal nuclease buffer [20 mM Tris–HCl (pH 7.6), 100 mM KCl, 0.3 M sucrose, 0.1% Triton X-100, 2 mM MgCl2 , 1 mM CaCl2 and cOmplete™ EDTA-free Protease Inhibitor Cocktail (Roche)] and digested with MNase at 25°C for 10 min.

Article Title: TRAF4 Is a Novel Phosphoinositide-Binding Protein Modulating Tight Junctions and Favoring Cell Migration
Article Snippet: The supernatant was incubated with 1 ml of His-Select Nickel Affinity Gel (Sigma) overnight at 4°C under agitation. .. Eluted fractions were dialyzed against Calmodulin Exchange buffer (10 mM Tris-HCl, 150 mM NaCl, 1 mM Mg(CH3 COO)2 , 1 mM imidazole, 10 mM β-mercaptoethanol, 0.1% NP40, 2 mM CaCl2 , complete protease inhibitor (Roche) [pH 8]).

Infection:

Article Title: Single-cell tracking of flavivirus RNA uncovers species-specific interactions with the immune system dictating disease outcome
Article Snippet: Fetal liver was homogenized and incubated in digestion medium (HBSS with 0.1% collagenase IV (Sigma), 40 mM HEPES, 2 M CaCl2 and 2 U ml−1 DNAse I (Roche) for 30 min at 37 °C. .. Expression of human CD90, CD38, CD45RA was assessed among the CD34+ population.

Expressing:

Article Title: Selective expansion of myeloid and NK cells in humanized mice yields human-like vaccine responses
Article Snippet: Fetal liver was homogenized and incubated in digestion medium (HBSS) with 0.1% collagenase IV (Sigma-Aldrich, Darmstadt, Germany), 40 mM HEPES, 2 M CaCl2 , and 2 U/ml DNAse I (Roche, Basel, Switzerland) for 30 min at 37 °C. .. Purification of human CD34+ cells were assessed by quantifying by flow cytometry using an anti-human CD34+-FITC antibody (dilution 1/100, clone 581, BD Biosciences, Franklin Lakes, NJ).

Article Title: Single-cell tracking of flavivirus RNA uncovers species-specific interactions with the immune system dictating disease outcome
Article Snippet: Fetal liver was homogenized and incubated in digestion medium (HBSS with 0.1% collagenase IV (Sigma), 40 mM HEPES, 2 M CaCl2 and 2 U ml−1 DNAse I (Roche) for 30 min at 37 °C. .. Fetal liver was homogenized and incubated in digestion medium (HBSS with 0.1% collagenase IV (Sigma), 40 mM HEPES, 2 M CaCl2 and 2 U ml−1 DNAse I (Roche) for 30 min at 37 °C.

Article Title: Regulation of HLTF-mediated PCNA polyubiquitination by RFC and PCNA monoubiquitination levels determines choice of damage tolerance pathway
Article Snippet: Then, stable cell lines expressing 3× FLAG-tagged HLTF (HLTF −/− /3xFLAG HLTF ) and a vector-transfected cell line (HLTF −/− /vector) were established. .. The precipitates (insoluble fraction) were suspended in micrococcal nuclease buffer [20 mM Tris–HCl (pH 7.6), 100 mM KCl, 0.3 M sucrose, 0.1% Triton X-100, 2 mM MgCl2 , 1 mM CaCl2 and cOmplete™ EDTA-free Protease Inhibitor Cocktail (Roche)] and digested with MNase at 25°C for 10 min.

BIA-KA:

Article Title: Neutron-encoded mass signatures for multi-plexed proteome quantification
Article Snippet: The cell pellets were resuspended in 8 M urea, 50 mM Tris pH 8.0, 5 mM CaCl2 , and protease inhibitors (Roche). .. The cell pellets were resuspended in 8 M urea, 50 mM Tris pH 8.0, 5 mM CaCl2 , and protease inhibitors (Roche).

Modification:

Article Title: Regulation of HLTF-mediated PCNA polyubiquitination by RFC and PCNA monoubiquitination levels determines choice of damage tolerance pathway
Article Snippet: The cell lines were maintained in Dulbecco's modified Eagle's medium (Wako) supplemented with 10% fetal bovine serum, 1× penicillin-streptomycin solution (Nacalai Tesque), and 0.4 mg/ml G418 at 37°C with 5% CO2 . .. The precipitates (insoluble fraction) were suspended in micrococcal nuclease buffer [20 mM Tris–HCl (pH 7.6), 100 mM KCl, 0.3 M sucrose, 0.1% Triton X-100, 2 mM MgCl2 , 1 mM CaCl2 and cOmplete™ EDTA-free Protease Inhibitor Cocktail (Roche)] and digested with MNase at 25°C for 10 min.

Western Blot:

Article Title: Early preclinical detection of prions in the skin of prion-infected animals
Article Snippet: The samples were carefully cleaned in Tris-buffered saline (TBS) containing 10 mM Tris-HCl, 133 mM NaCl, pH 7.4 for three times before processing of each specimen in order to avoid blood contamination. .. For western blotting, or sPMCA assay, the skin samples were incubated in TBS containing 2 mM CaCl2 and 0.25% (10% w/v) collagenase A (Roche) in a shaker at 37 °C for 4 h. Beads Beater was used to make tissue homogenates and the samples were then centrifuged at 500×g for 5 min. .. The supernatant (S1) was transferred to a clean tube for future use and discard the pellet (P1).

Article Title: Regulation of HLTF-mediated PCNA polyubiquitination by RFC and PCNA monoubiquitination levels determines choice of damage tolerance pathway
Article Snippet: Mutations in both alleles (one and two base deletions) as well as no HLTF signal by western blotting were confirmed (data not shown). .. The precipitates (insoluble fraction) were suspended in micrococcal nuclease buffer [20 mM Tris–HCl (pH 7.6), 100 mM KCl, 0.3 M sucrose, 0.1% Triton X-100, 2 mM MgCl2 , 1 mM CaCl2 and cOmplete™ EDTA-free Protease Inhibitor Cocktail (Roche)] and digested with MNase at 25°C for 10 min.

Article Title: TRAF4 Is a Novel Phosphoinositide-Binding Protein Modulating Tight Junctions and Favoring Cell Migration
Article Snippet: Eluted fractions were dialyzed against Calmodulin Exchange buffer (10 mM Tris-HCl, 150 mM NaCl, 1 mM Mg(CH3 COO)2 , 1 mM imidazole, 10 mM β-mercaptoethanol, 0.1% NP40, 2 mM CaCl2 , complete protease inhibitor (Roche) [pH 8]). .. Eluted fractions were dialyzed against Calmodulin Exchange buffer (10 mM Tris-HCl, 150 mM NaCl, 1 mM Mg(CH3 COO)2 , 1 mM imidazole, 10 mM β-mercaptoethanol, 0.1% NP40, 2 mM CaCl2 , complete protease inhibitor (Roche) [pH 8]).

Over Expression:

Article Title: Role of Multidrug Resistance–Associated Protein 4 in the Basolateral Efflux of Hepatically Derived Enalaprilat
Article Snippet: Paragraph title: Human Embryonic Kidney Cell Culture and Overexpression of MRP3 and MRP4. ... The final cell pellet was overlaid with 10 ml TSB containing 0.25 mM CaCl2 and protease inhibitors (complete Mini EDTA-free; Roche Diagnostics), snap-frozen in liquid nitrogen, and stored at −80°C.

Transfection:

Article Title: Role of Multidrug Resistance–Associated Protein 4 in the Basolateral Efflux of Hepatically Derived Enalaprilat
Article Snippet: For MRP3, transient transfection of HEK293T cells with X-tremeGENE 9 DNA Transfection Reagent (Roche Diagnostics, Mannheim, Germany) was performed according to the manufacturer’s instructions. .. The final cell pellet was overlaid with 10 ml TSB containing 0.25 mM CaCl2 and protease inhibitors (complete Mini EDTA-free; Roche Diagnostics), snap-frozen in liquid nitrogen, and stored at −80°C.

Serial Dilution:

Article Title: Early preclinical detection of prions in the skin of prion-infected animals
Article Snippet: For western blotting, or sPMCA assay, the skin samples were incubated in TBS containing 2 mM CaCl2 and 0.25% (10% w/v) collagenase A (Roche) in a shaker at 37 °C for 4 h. Beads Beater was used to make tissue homogenates and the samples were then centrifuged at 500×g for 5 min. .. For western blotting, or sPMCA assay, the skin samples were incubated in TBS containing 2 mM CaCl2 and 0.25% (10% w/v) collagenase A (Roche) in a shaker at 37 °C for 4 h. Beads Beater was used to make tissue homogenates and the samples were then centrifuged at 500×g for 5 min.

Cell Culture:

Article Title: Role of Multidrug Resistance–Associated Protein 4 in the Basolateral Efflux of Hepatically Derived Enalaprilat
Article Snippet: Paragraph title: Human Embryonic Kidney Cell Culture and Overexpression of MRP3 and MRP4. ... The final cell pellet was overlaid with 10 ml TSB containing 0.25 mM CaCl2 and protease inhibitors (complete Mini EDTA-free; Roche Diagnostics), snap-frozen in liquid nitrogen, and stored at −80°C.

Light Microscopy:

Article Title: A switch in surface polymer biogenesis triggers growth-phase-dependent and antibiotic-induced bacteriolysis
Article Snippet: Complete protoplasting was monitored by light microscopy. .. Protoplasts were pelleted by centrifugation at 5000 g for 5 min and resuspended in 2 ml cold hypotonic buffer to lyse them (20 mM HEPES (Na+ ), pH 8.0, 100 mM NaCl, 1 mM dithiothreitol (DTT), 1 mM MgCl2 , 1 mM CaCl2 , 2X complete protease inhibitors (Roche), 6 μg/ml RNAse A, 6 μg/ml DNAse).

Generated:

Article Title: A switch in surface polymer biogenesis triggers growth-phase-dependent and antibiotic-induced bacteriolysis
Article Snippet: Protoplasts were generated by addition of 20 mg/ml lysozyme and 100 units mutanolysin (Sigma) and incubated at 37°C for 30 min. .. Protoplasts were pelleted by centrifugation at 5000 g for 5 min and resuspended in 2 ml cold hypotonic buffer to lyse them (20 mM HEPES (Na+ ), pH 8.0, 100 mM NaCl, 1 mM dithiothreitol (DTT), 1 mM MgCl2 , 1 mM CaCl2 , 2X complete protease inhibitors (Roche), 6 μg/ml RNAse A, 6 μg/ml DNAse).

Confocal Laser Scanning Microscopy:

Article Title: EGF receptor kinase suppresses ciliogenesis through activation of USP8 deubiquitinase
Article Snippet: The embryo or fishes samples were soaked in a cacodylate-buffered solution containing 2 mM CaCl2 , 30% sucrose, and protease inhibitor cocktail tablets (Roche Diagnostics, Mannheim, Germany) at 4 ℃ for overnight. .. The embryo or fishes samples were soaked in a cacodylate-buffered solution containing 2 mM CaCl2 , 30% sucrose, and protease inhibitor cocktail tablets (Roche Diagnostics, Mannheim, Germany) at 4 ℃ for overnight.

Imaging:

Article Title: Spermine synthase deficiency causes lysosomal dysfunction and oxidative stress in models of Snyder-Robinson syndrome
Article Snippet: For immunoblot analysis of cathepsins, tissues were homogenized on ice in lysis buffer containing 20 mM HEPES, 100 mM NaCl, 1 mM CaCl2 , 0.5% Triton X-100 and cOmplete protease inhibitor cocktail (Roche), followed by 10 min centrifugation at 10,000×g at 4 °C. .. After blocking, the membrane was incubated with primary antibodies overnight at 4 °C and near infrared dye-conjugated secondary antibodies for 2 h at room temperature.

Protein Concentration:

Article Title: Neutron-encoded mass signatures for multi-plexed proteome quantification
Article Snippet: The cell pellets were resuspended in 8 M urea, 50 mM Tris pH 8.0, 5 mM CaCl2 , and protease inhibitors (Roche). .. The cell pellets were resuspended in 8 M urea, 50 mM Tris pH 8.0, 5 mM CaCl2 , and protease inhibitors (Roche).

Sonication:

Article Title: TRAF4 Is a Novel Phosphoinositide-Binding Protein Modulating Tight Junctions and Favoring Cell Migration
Article Snippet: The cell pellet was lysed and sonicated in 50 ml of Lysis buffer (50 mM NaH2 PO4 /Na2 HPO4 , 300 mM NaCl, 10 mM imidazole [pH 8.0]) containing EDTA-free complete protease inhibitor tablets (Roche). .. Eluted fractions were dialyzed against Calmodulin Exchange buffer (10 mM Tris-HCl, 150 mM NaCl, 1 mM Mg(CH3 COO)2 , 1 mM imidazole, 10 mM β-mercaptoethanol, 0.1% NP40, 2 mM CaCl2 , complete protease inhibitor (Roche) [pH 8]).

Injection:

Article Title: Copper amine oxidases catalyze the oxidative deamination and hydrolysis of cyclic imines
Article Snippet: The derivatized enzyme was digested in a reaction mixture comprising 100 mM Tris-HCl (pH 8.0), 10 mM CaCl2 , and 25 μg chymotrypsin (Roche Diagnostics, Basel, Schweiz). .. The LC/MS conditions were as follows: flow rate, 1 mL min−1 ; solvent A, 0.05% (v/v) formic acid; solvent B, acetonitrile; and column, COSMOSIL πNAP 4.6 × 150 (Nacalai Tesque).

Recombinant:

Article Title: TRAF4 Is a Novel Phosphoinositide-Binding Protein Modulating Tight Junctions and Favoring Cell Migration
Article Snippet: Paragraph title: Production and Purification of Recombinant Proteins ... Eluted fractions were dialyzed against Calmodulin Exchange buffer (10 mM Tris-HCl, 150 mM NaCl, 1 mM Mg(CH3 COO)2 , 1 mM imidazole, 10 mM β-mercaptoethanol, 0.1% NP40, 2 mM CaCl2 , complete protease inhibitor (Roche) [pH 8]).

Immunofluorescence:

Article Title: Distinct Regulatory Mechanisms Govern Embryonic versus Adult Adipocyte Maturation
Article Snippet: Briefly, dissected sWAT from two mice of each genotype were digested for 1 hour at 37 degree in PBS containing 10 mM CaCl2 , 2.4 units/mL dispase II (Roche Diagnostics Corporation, Indianapolis, IN) and 1.5 units/mL collagenase D (Roche Diagnostics Corporation, Indianapolis, IN). .. Briefly, dissected sWAT from two mice of each genotype were digested for 1 hour at 37 degree in PBS containing 10 mM CaCl2 , 2.4 units/mL dispase II (Roche Diagnostics Corporation, Indianapolis, IN) and 1.5 units/mL collagenase D (Roche Diagnostics Corporation, Indianapolis, IN).

Isolation:

Article Title: Selective expansion of myeloid and NK cells in humanized mice yields human-like vaccine responses
Article Snippet: Paragraph title: Isolation of human CD34+ HSC ... Fetal liver was homogenized and incubated in digestion medium (HBSS) with 0.1% collagenase IV (Sigma-Aldrich, Darmstadt, Germany), 40 mM HEPES, 2 M CaCl2 , and 2 U/ml DNAse I (Roche, Basel, Switzerland) for 30 min at 37 °C.

Article Title: A switch in surface polymer biogenesis triggers growth-phase-dependent and antibiotic-induced bacteriolysis
Article Snippet: Paragraph title: Isolation and analysis of pneumococcal LTAs ... Protoplasts were pelleted by centrifugation at 5000 g for 5 min and resuspended in 2 ml cold hypotonic buffer to lyse them (20 mM HEPES (Na+ ), pH 8.0, 100 mM NaCl, 1 mM dithiothreitol (DTT), 1 mM MgCl2 , 1 mM CaCl2 , 2X complete protease inhibitors (Roche), 6 μg/ml RNAse A, 6 μg/ml DNAse).

Article Title: Single-cell tracking of flavivirus RNA uncovers species-specific interactions with the immune system dictating disease outcome
Article Snippet: Paragraph title: Isolation of human CD34+ and murine CD117+ HSC ... Fetal liver was homogenized and incubated in digestion medium (HBSS with 0.1% collagenase IV (Sigma), 40 mM HEPES, 2 M CaCl2 and 2 U ml−1 DNAse I (Roche) for 30 min at 37 °C.

Article Title: Altenusin, a Nonsteroidal Microbial Metabolite, Attenuates Nonalcoholic Fatty Liver Disease by Activating the Farnesoid X Receptor
Article Snippet: Paragraph title: Isolation and Culture of Primary Mouse Hepatocytes. ... Briefly, the liver was first perfused with Hanks’ buffered salt solution containing 0.5 mM EGTA and 0.1 M HEPES at 5 ml/min for 5–10 minutes and then perfused with L-15 medium containing 1.8 mM CaCl2 , 0.1 M HEPES, and 20 μg/ml liberase (Roche, Indianapolis, IN).

Article Title: Elevated Expression of Osteopontin May Be Related to Adipose Tissue Macrophage Accumulation and Liver Steatosis in Morbid Obesity
Article Snippet: Paragraph title: Isolation of hepatocytes and nonparenchymal fraction from liver. ... Mouse livers were perfused first with a HEPES buffer containing 8 g/l NaCl, 33 mg/l Na2 HPO4 , 200 mg/l KCl, and 2.38 g/l HEPES, pH 7.65, for 2 min at 5 ml/min and then with HEPES buffer supplemented with 1.5 g/l CaCl2 and 25 mg/l liberase (Roche Diagnostics) for 7 min at 5 ml/min.

Article Title: Optogenetic stimulation of Gs-signaling in the heart with high spatio-temporal precision
Article Snippet: Paragraph title: Isolation of adult ventricular cardiomyocytes ... Briefly, hearts were perfused in the Langendorff configuration with dissociation solution (in mM: 135 NaCl, 4 KCl, 1 MgCl2 , 2.5 HEPES, 5 glucose, 25 butanedione monoxime; pH 7.4) for 5 min at 37 °C and then 50 μM CaCl2 , 0.8 mg mL−1 collagenase B (Roche) and 0.3 mg mL−1 trypsin (Invitrogen) was added for 12–13 min. Ventricles were cut in small pieces and mechanically dissected, cells were filtered through a nylon mesh and the pellet was resuspended in dissociation solution with 50 μM CaCl2 , 5% FCS but without butanedione monoxime.

Flow Cytometry:

Article Title: Selective expansion of myeloid and NK cells in humanized mice yields human-like vaccine responses
Article Snippet: Fetal liver was homogenized and incubated in digestion medium (HBSS) with 0.1% collagenase IV (Sigma-Aldrich, Darmstadt, Germany), 40 mM HEPES, 2 M CaCl2 , and 2 U/ml DNAse I (Roche, Basel, Switzerland) for 30 min at 37 °C. .. Human CD34+ HSC were isolated using a CD34+ HSC isolation kit (Stem Cell Technologies, Vancouver, British Columbia, Canada) according to the manufacturer’ protocol.

Article Title: Single-cell tracking of flavivirus RNA uncovers species-specific interactions with the immune system dictating disease outcome
Article Snippet: Fetal liver was homogenized and incubated in digestion medium (HBSS with 0.1% collagenase IV (Sigma), 40 mM HEPES, 2 M CaCl2 and 2 U ml−1 DNAse I (Roche) for 30 min at 37 °C. .. Human CD34+ HSC were isolated using a CD34+ HSC isolation kit (Stem Cell Technologies) according to the manufacturer' protocol.

Article Title: Copper amine oxidases catalyze the oxidative deamination and hydrolysis of cyclic imines
Article Snippet: The derivatized enzyme was digested in a reaction mixture comprising 100 mM Tris-HCl (pH 8.0), 10 mM CaCl2 , and 25 μg chymotrypsin (Roche Diagnostics, Basel, Schweiz). .. The derivatized enzyme was digested in a reaction mixture comprising 100 mM Tris-HCl (pH 8.0), 10 mM CaCl2 , and 25 μg chymotrypsin (Roche Diagnostics, Basel, Schweiz).

Article Title: Distinct Regulatory Mechanisms Govern Embryonic versus Adult Adipocyte Maturation
Article Snippet: Briefly, dissected sWAT from two mice of each genotype were digested for 1 hour at 37 degree in PBS containing 10 mM CaCl2 , 2.4 units/mL dispase II (Roche Diagnostics Corporation, Indianapolis, IN) and 1.5 units/mL collagenase D (Roche Diagnostics Corporation, Indianapolis, IN). .. Briefly, dissected sWAT from two mice of each genotype were digested for 1 hour at 37 degree in PBS containing 10 mM CaCl2 , 2.4 units/mL dispase II (Roche Diagnostics Corporation, Indianapolis, IN) and 1.5 units/mL collagenase D (Roche Diagnostics Corporation, Indianapolis, IN).

Purification:

Article Title: Selective expansion of myeloid and NK cells in humanized mice yields human-like vaccine responses
Article Snippet: Fetal liver was homogenized and incubated in digestion medium (HBSS) with 0.1% collagenase IV (Sigma-Aldrich, Darmstadt, Germany), 40 mM HEPES, 2 M CaCl2 , and 2 U/ml DNAse I (Roche, Basel, Switzerland) for 30 min at 37 °C. .. Human CD34+ HSC were isolated using a CD34+ HSC isolation kit (Stem Cell Technologies, Vancouver, British Columbia, Canada) according to the manufacturer’ protocol.

Article Title: Single-cell tracking of flavivirus RNA uncovers species-specific interactions with the immune system dictating disease outcome
Article Snippet: Fetal liver was homogenized and incubated in digestion medium (HBSS with 0.1% collagenase IV (Sigma), 40 mM HEPES, 2 M CaCl2 and 2 U ml−1 DNAse I (Roche) for 30 min at 37 °C. .. Human CD34+ HSC were isolated using a CD34+ HSC isolation kit (Stem Cell Technologies) according to the manufacturer' protocol.

Article Title: TRAF4 Is a Novel Phosphoinositide-Binding Protein Modulating Tight Junctions and Favoring Cell Migration
Article Snippet: Paragraph title: Production and Purification of Recombinant Proteins ... Eluted fractions were dialyzed against Calmodulin Exchange buffer (10 mM Tris-HCl, 150 mM NaCl, 1 mM Mg(CH3 COO)2 , 1 mM imidazole, 10 mM β-mercaptoethanol, 0.1% NP40, 2 mM CaCl2 , complete protease inhibitor (Roche) [pH 8]).

Polyacrylamide Gel Electrophoresis:

Article Title: A switch in surface polymer biogenesis triggers growth-phase-dependent and antibiotic-induced bacteriolysis
Article Snippet: Protoplasts were pelleted by centrifugation at 5000 g for 5 min and resuspended in 2 ml cold hypotonic buffer to lyse them (20 mM HEPES (Na+ ), pH 8.0, 100 mM NaCl, 1 mM dithiothreitol (DTT), 1 mM MgCl2 , 1 mM CaCl2 , 2X complete protease inhibitors (Roche), 6 μg/ml RNAse A, 6 μg/ml DNAse). .. Unbroken spheroplasts were removed by centrifugation 5,000 rpm for 10 min, and then the membrane fraction was collected by ultracentrifugation at 100,000 g for 1 hr at 4°C.

Lysis:

Article Title: Neutron-encoded mass signatures for multi-plexed proteome quantification
Article Snippet: Cell pellets were re-suspended in 5mL lysis buffer (50mM Tris pH8, 8M urea, 75mM sodium chloride, 100mM sodium butyrate, 1mM sodium orthovanadate, protease and phosphatase inhibitor tablet), and total protein was extracted by glass bead milling (Retsch). .. The cell pellets were resuspended in 8 M urea, 50 mM Tris pH 8.0, 5 mM CaCl2 , and protease inhibitors (Roche).

Article Title: Spermine synthase deficiency causes lysosomal dysfunction and oxidative stress in models of Snyder-Robinson syndrome
Article Snippet: Specifically, histogram plots in Fig. and Supplementary Fig. are analyzed using ImageJ Plot Profile and surface plots in Fig. and Supplementary Fig. are plotted using ImageJ Interactive 3D Surface Plot plugin. .. For immunoblot analysis of cathepsins, tissues were homogenized on ice in lysis buffer containing 20 mM HEPES, 100 mM NaCl, 1 mM CaCl2 , 0.5% Triton X-100 and cOmplete protease inhibitor cocktail (Roche), followed by 10 min centrifugation at 10,000×g at 4 °C. .. The supernatants were then mixed with Laemmli sample buffer and heated at 95 °C for 10 min. Proteins were separated on a Bis-Tris 10% gel and transferred to a nitrocellulose membrane.

Article Title: Regulation of HLTF-mediated PCNA polyubiquitination by RFC and PCNA monoubiquitination levels determines choice of damage tolerance pathway
Article Snippet: The cells were suspended in lysis buffer [20 mM Tris–HCl (pH 7.6), 150 mM KCl, 10% glycerol, 0.5% NP-40, 1.5 mM MgCl2 , 0.2 μg/ml antipain, 0.2 μg/ml aprotinin, 0.1 μg/ml leupeptin, 0.08 μg/ml pepstatin, 0.05 mM EGTA and 0.25 mM phenylmethylsulfonyl fluoride (PMSF)], and soluble materials were separated by centrifugation. .. The precipitates (insoluble fraction) were suspended in micrococcal nuclease buffer [20 mM Tris–HCl (pH 7.6), 100 mM KCl, 0.3 M sucrose, 0.1% Triton X-100, 2 mM MgCl2 , 1 mM CaCl2 and cOmplete™ EDTA-free Protease Inhibitor Cocktail (Roche)] and digested with MNase at 25°C for 10 min.

Article Title: TRAF4 Is a Novel Phosphoinositide-Binding Protein Modulating Tight Junctions and Favoring Cell Migration
Article Snippet: The cell pellet was lysed and sonicated in 50 ml of Lysis buffer (50 mM NaH2 PO4 /Na2 HPO4 , 300 mM NaCl, 10 mM imidazole [pH 8.0]) containing EDTA-free complete protease inhibitor tablets (Roche). .. Eluted fractions were dialyzed against Calmodulin Exchange buffer (10 mM Tris-HCl, 150 mM NaCl, 1 mM Mg(CH3 COO)2 , 1 mM imidazole, 10 mM β-mercaptoethanol, 0.1% NP40, 2 mM CaCl2 , complete protease inhibitor (Roche) [pH 8]).

Mouse Assay:

Article Title: Early preclinical detection of prions in the skin of prion-infected animals
Article Snippet: Skin samples at 0.5 × 0.5 cm from ear pinna, thigh, back, belly, or rib were collected from hamsters inoculated with 263K scrapie, Tg40h mice, or PBS-negative controls, respectively. .. For western blotting, or sPMCA assay, the skin samples were incubated in TBS containing 2 mM CaCl2 and 0.25% (10% w/v) collagenase A (Roche) in a shaker at 37 °C for 4 h. Beads Beater was used to make tissue homogenates and the samples were then centrifuged at 500×g for 5 min.

Article Title: Altenusin, a Nonsteroidal Microbial Metabolite, Attenuates Nonalcoholic Fatty Liver Disease by Activating the Farnesoid X Receptor
Article Snippet: Primary mouse hepatocytes were isolated from 6- to 8-week-old male mice, as previously described ( ). .. Briefly, the liver was first perfused with Hanks’ buffered salt solution containing 0.5 mM EGTA and 0.1 M HEPES at 5 ml/min for 5–10 minutes and then perfused with L-15 medium containing 1.8 mM CaCl2 , 0.1 M HEPES, and 20 μg/ml liberase (Roche, Indianapolis, IN).

Article Title: Distinct Regulatory Mechanisms Govern Embryonic versus Adult Adipocyte Maturation
Article Snippet: SVF Culture and Adipocyte Differentiation SVF fractions from sWAT were obtained from 5 weeks old male mice of each genotype as indicated. .. Briefly, dissected sWAT from two mice of each genotype were digested for 1 hour at 37 degree in PBS containing 10 mM CaCl2 , 2.4 units/mL dispase II (Roche Diagnostics Corporation, Indianapolis, IN) and 1.5 units/mL collagenase D (Roche Diagnostics Corporation, Indianapolis, IN). .. Digested cell/tissue mixture was filtered through a 100 μm cell strainer to remove undigested tissues.

Article Title: Optogenetic stimulation of Gs-signaling in the heart with high spatio-temporal precision
Article Snippet: Ventricular cardiomyocytes were isolated from JellyOp mice, as described previously . .. Briefly, hearts were perfused in the Langendorff configuration with dissociation solution (in mM: 135 NaCl, 4 KCl, 1 MgCl2 , 2.5 HEPES, 5 glucose, 25 butanedione monoxime; pH 7.4) for 5 min at 37 °C and then 50 μM CaCl2 , 0.8 mg mL−1 collagenase B (Roche) and 0.3 mg mL−1 trypsin (Invitrogen) was added for 12–13 min. Ventricles were cut in small pieces and mechanically dissected, cells were filtered through a nylon mesh and the pellet was resuspended in dissociation solution with 50 μM CaCl2 , 5% FCS but without butanedione monoxime.

SDS Page:

Article Title: TRAF4 Is a Novel Phosphoinositide-Binding Protein Modulating Tight Junctions and Favoring Cell Migration
Article Snippet: Eluted fractions were dialyzed against Calmodulin Exchange buffer (10 mM Tris-HCl, 150 mM NaCl, 1 mM Mg(CH3 COO)2 , 1 mM imidazole, 10 mM β-mercaptoethanol, 0.1% NP40, 2 mM CaCl2 , complete protease inhibitor (Roche) [pH 8]). .. Eluted fractions were dialyzed against Calmodulin Exchange buffer (10 mM Tris-HCl, 150 mM NaCl, 1 mM Mg(CH3 COO)2 , 1 mM imidazole, 10 mM β-mercaptoethanol, 0.1% NP40, 2 mM CaCl2 , complete protease inhibitor (Roche) [pH 8]).

Plasmid Preparation:

Article Title: Regulation of HLTF-mediated PCNA polyubiquitination by RFC and PCNA monoubiquitination levels determines choice of damage tolerance pathway
Article Snippet: Then, stable cell lines expressing 3× FLAG-tagged HLTF (HLTF −/− /3xFLAG HLTF ) and a vector-transfected cell line (HLTF −/− /vector) were established. .. The precipitates (insoluble fraction) were suspended in micrococcal nuclease buffer [20 mM Tris–HCl (pH 7.6), 100 mM KCl, 0.3 M sucrose, 0.1% Triton X-100, 2 mM MgCl2 , 1 mM CaCl2 and cOmplete™ EDTA-free Protease Inhibitor Cocktail (Roche)] and digested with MNase at 25°C for 10 min.

Software:

Article Title: Spermine synthase deficiency causes lysosomal dysfunction and oxidative stress in models of Snyder-Robinson syndrome
Article Snippet: For immunoblot analysis of cathepsins, tissues were homogenized on ice in lysis buffer containing 20 mM HEPES, 100 mM NaCl, 1 mM CaCl2 , 0.5% Triton X-100 and cOmplete protease inhibitor cocktail (Roche), followed by 10 min centrifugation at 10,000×g at 4 °C. .. After blocking, the membrane was incubated with primary antibodies overnight at 4 °C and near infrared dye-conjugated secondary antibodies for 2 h at room temperature.

Irradiation:

Article Title: Regulation of HLTF-mediated PCNA polyubiquitination by RFC and PCNA monoubiquitination levels determines choice of damage tolerance pathway
Article Snippet: After one wash with phosphate-buffered saline, cells were irradiated with UVC (15 J/m2 ), incubated under the above-described conditions for 3 h, and harvested. .. The precipitates (insoluble fraction) were suspended in micrococcal nuclease buffer [20 mM Tris–HCl (pH 7.6), 100 mM KCl, 0.3 M sucrose, 0.1% Triton X-100, 2 mM MgCl2 , 1 mM CaCl2 and cOmplete™ EDTA-free Protease Inhibitor Cocktail (Roche)] and digested with MNase at 25°C for 10 min.

RNA Extraction:

Article Title: Elevated Expression of Osteopontin May Be Related to Adipose Tissue Macrophage Accumulation and Liver Steatosis in Morbid Obesity
Article Snippet: Mouse livers were perfused first with a HEPES buffer containing 8 g/l NaCl, 33 mg/l Na2 HPO4 , 200 mg/l KCl, and 2.38 g/l HEPES, pH 7.65, for 2 min at 5 ml/min and then with HEPES buffer supplemented with 1.5 g/l CaCl2 and 25 mg/l liberase (Roche Diagnostics) for 7 min at 5 ml/min. .. Mouse livers were perfused first with a HEPES buffer containing 8 g/l NaCl, 33 mg/l Na2 HPO4 , 200 mg/l KCl, and 2.38 g/l HEPES, pH 7.65, for 2 min at 5 ml/min and then with HEPES buffer supplemented with 1.5 g/l CaCl2 and 25 mg/l liberase (Roche Diagnostics) for 7 min at 5 ml/min.

Article Title: Distinct Regulatory Mechanisms Govern Embryonic versus Adult Adipocyte Maturation
Article Snippet: Briefly, dissected sWAT from two mice of each genotype were digested for 1 hour at 37 degree in PBS containing 10 mM CaCl2 , 2.4 units/mL dispase II (Roche Diagnostics Corporation, Indianapolis, IN) and 1.5 units/mL collagenase D (Roche Diagnostics Corporation, Indianapolis, IN). .. Briefly, dissected sWAT from two mice of each genotype were digested for 1 hour at 37 degree in PBS containing 10 mM CaCl2 , 2.4 units/mL dispase II (Roche Diagnostics Corporation, Indianapolis, IN) and 1.5 units/mL collagenase D (Roche Diagnostics Corporation, Indianapolis, IN).

Sample Prep:

Article Title: Neutron-encoded mass signatures for multi-plexed proteome quantification
Article Snippet: Paragraph title: Sample preparation ... The cell pellets were resuspended in 8 M urea, 50 mM Tris pH 8.0, 5 mM CaCl2 , and protease inhibitors (Roche).

Homogenization:

Article Title: The Immune Interplay between Thyroid Papillary Carcinoma and Hepatic Fibrosis
Article Snippet: Immunoblot analysis of α smooth muscle actin (αSMA) in liver extracts was performed with modifications as previously described [ ]. .. Whole liver protein extracts were prepared in liver homogenization buffer (50 mmol/L Tris–HCl [pH 7.6], 0.25% Triton-X 100, 0.15 M NaCl, 10 mM CaCl2 and complete mini EDTA-free protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany)). .. Next, proteins (30 μg per lane) were resolved on a 10% (wt/vol) SDS-polyacrylamide gel (Novex, Groningen, The Netherlands) under reducing conditions.

Fractionation:

Article Title: Alzheimer Amyloid-? Oligomer Bound to Post-Synaptic Prion Protein Activates Fyn to Impair Neurons
Article Snippet: Paragraph title: Subcellular Fractionation of Brain Tissue ... Rat forebrains were homogenized in ice-cold 5 mM NaHEPES, pH7.4, 1 mM MgCl2 , 0.5 mM CaCl2 , complete protease inhibitor cocktail (Roche), phosphatase inhibitor (Roche) with a glass/Teflon pestel.

Liquid Chromatography with Mass Spectroscopy:

Article Title: Copper amine oxidases catalyze the oxidative deamination and hydrolysis of cyclic imines
Article Snippet: The derivatized enzyme was digested in a reaction mixture comprising 100 mM Tris-HCl (pH 8.0), 10 mM CaCl2 , and 25 μg chymotrypsin (Roche Diagnostics, Basel, Schweiz). .. The derivatized enzyme was digested in a reaction mixture comprising 100 mM Tris-HCl (pH 8.0), 10 mM CaCl2 , and 25 μg chymotrypsin (Roche Diagnostics, Basel, Schweiz).

Protease Inhibitor:

Article Title: Early preclinical detection of prions in the skin of prion-infected animals
Article Snippet: For western blotting, or sPMCA assay, the skin samples were incubated in TBS containing 2 mM CaCl2 and 0.25% (10% w/v) collagenase A (Roche) in a shaker at 37 °C for 4 h. Beads Beater was used to make tissue homogenates and the samples were then centrifuged at 500×g for 5 min. .. For western blotting, or sPMCA assay, the skin samples were incubated in TBS containing 2 mM CaCl2 and 0.25% (10% w/v) collagenase A (Roche) in a shaker at 37 °C for 4 h. Beads Beater was used to make tissue homogenates and the samples were then centrifuged at 500×g for 5 min.

Article Title: The Immune Interplay between Thyroid Papillary Carcinoma and Hepatic Fibrosis
Article Snippet: Immunoblot analysis of α smooth muscle actin (αSMA) in liver extracts was performed with modifications as previously described [ ]. .. Whole liver protein extracts were prepared in liver homogenization buffer (50 mmol/L Tris–HCl [pH 7.6], 0.25% Triton-X 100, 0.15 M NaCl, 10 mM CaCl2 and complete mini EDTA-free protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany)). .. Next, proteins (30 μg per lane) were resolved on a 10% (wt/vol) SDS-polyacrylamide gel (Novex, Groningen, The Netherlands) under reducing conditions.

Article Title: EGF receptor kinase suppresses ciliogenesis through activation of USP8 deubiquitinase
Article Snippet: Zebrafish at 27 hpf and 4 dpf zebrafish were fixed in a 4% paraformaldehyde/cacodylate-buffered solution (0.15 M, pH 7.4) containing 2 mM CaCl2 for overnight at 4 °C. .. The embryo or fishes samples were soaked in a cacodylate-buffered solution containing 2 mM CaCl2 , 30% sucrose, and protease inhibitor cocktail tablets (Roche Diagnostics, Mannheim, Germany) at 4 ℃ for overnight. .. Then, the samples were quickly frozen in OCT compound (Sakura Finetek, Tokyo, Japan) and 12-μm thickness serial cryostat sections were cut and mounted on glass slides, then quickly blocked by 0.1 M phosphate buffer (pH 7.4) containing 4% Block Ace (DS Pharma Biomedical), protease cocktail, and 0.02% saponin, and incubated at room temperature (RT) for 20 min. Then, the samples were incubated at 4 ℃ for 1 overnight for primary Abs, and the 2 h at RT for secondary Abs.

Article Title: Alzheimer Amyloid-? Oligomer Bound to Post-Synaptic Prion Protein Activates Fyn to Impair Neurons
Article Snippet: Values were collected from at least 4 fields in each culture condition. .. Rat forebrains were homogenized in ice-cold 5 mM NaHEPES, pH7.4, 1 mM MgCl2 , 0.5 mM CaCl2 , complete protease inhibitor cocktail (Roche), phosphatase inhibitor (Roche) with a glass/Teflon pestel. .. Extract was spun at 1400 × g for 10 min. Supernatant (S1) was centrifuged at 13,800 × g for 10 min to collect the pellet (P2).

Article Title: Spermine synthase deficiency causes lysosomal dysfunction and oxidative stress in models of Snyder-Robinson syndrome
Article Snippet: Specifically, histogram plots in Fig. and Supplementary Fig. are analyzed using ImageJ Plot Profile and surface plots in Fig. and Supplementary Fig. are plotted using ImageJ Interactive 3D Surface Plot plugin. .. For immunoblot analysis of cathepsins, tissues were homogenized on ice in lysis buffer containing 20 mM HEPES, 100 mM NaCl, 1 mM CaCl2 , 0.5% Triton X-100 and cOmplete protease inhibitor cocktail (Roche), followed by 10 min centrifugation at 10,000×g at 4 °C. .. The supernatants were then mixed with Laemmli sample buffer and heated at 95 °C for 10 min. Proteins were separated on a Bis-Tris 10% gel and transferred to a nitrocellulose membrane.

Article Title: Regulation of HLTF-mediated PCNA polyubiquitination by RFC and PCNA monoubiquitination levels determines choice of damage tolerance pathway
Article Snippet: The cells were suspended in lysis buffer [20 mM Tris–HCl (pH 7.6), 150 mM KCl, 10% glycerol, 0.5% NP-40, 1.5 mM MgCl2 , 0.2 μg/ml antipain, 0.2 μg/ml aprotinin, 0.1 μg/ml leupeptin, 0.08 μg/ml pepstatin, 0.05 mM EGTA and 0.25 mM phenylmethylsulfonyl fluoride (PMSF)], and soluble materials were separated by centrifugation. .. The precipitates (insoluble fraction) were suspended in micrococcal nuclease buffer [20 mM Tris–HCl (pH 7.6), 100 mM KCl, 0.3 M sucrose, 0.1% Triton X-100, 2 mM MgCl2 , 1 mM CaCl2 and cOmplete™ EDTA-free Protease Inhibitor Cocktail (Roche)] and digested with MNase at 25°C for 10 min. .. The reaction was terminated by addition of EDTA, and the MNase-soluble fraction (chromatin fraction) was collected by centrifugation.

Article Title: TRAF4 Is a Novel Phosphoinositide-Binding Protein Modulating Tight Junctions and Favoring Cell Migration
Article Snippet: Bound proteins were eluted with elution buffer (20 mM NaH2 PO4 /Na2 HPO4 , 250 mM imidazole [pH 7.4]). .. Eluted fractions were dialyzed against Calmodulin Exchange buffer (10 mM Tris-HCl, 150 mM NaCl, 1 mM Mg(CH3 COO)2 , 1 mM imidazole, 10 mM β-mercaptoethanol, 0.1% NP40, 2 mM CaCl2 , complete protease inhibitor (Roche) [pH 8]). .. Dialyzed fractions were incubated with 1 ml of Calmodulin Affinity Resin (Stratagene), supplemented with 3 µl of 2 M CaCl2 and then incubated overnight at 4°C under agitation.

Staining:

Article Title: Distinct Regulatory Mechanisms Govern Embryonic versus Adult Adipocyte Maturation
Article Snippet: Briefly, dissected sWAT from two mice of each genotype were digested for 1 hour at 37 degree in PBS containing 10 mM CaCl2 , 2.4 units/mL dispase II (Roche Diagnostics Corporation, Indianapolis, IN) and 1.5 units/mL collagenase D (Roche Diagnostics Corporation, Indianapolis, IN). .. Briefly, dissected sWAT from two mice of each genotype were digested for 1 hour at 37 degree in PBS containing 10 mM CaCl2 , 2.4 units/mL dispase II (Roche Diagnostics Corporation, Indianapolis, IN) and 1.5 units/mL collagenase D (Roche Diagnostics Corporation, Indianapolis, IN).

other:

Article Title: Prion Seeds Distribute throughout the Eyes of Sporadic Creutzfeldt-Jakob Disease Patients
Article Snippet: Eye homogenates (10% [wt/vol]) were prepared in PBS containing 2 mM CaCl2 and 0.25% (wt/vol) collagenase A (Roche).

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  • 99
    Roche proteinase k
    Halo of Lewy bodies is fuzzy and contains ubiquitin, whereas the core consists of compacted α-synuclein. Transmission electron microscopic images of Lewy bodies without ( top panel ) and after <t>proteinase</t> K digestion ( bottom panel ). Lewy bodies were affinity-isolated with anti-α-synuclein antiserum from brain cortex and immunostained with anti-ubiquitin and anti-synuclein primary and gold particle-labeled secondary antibodies and counterstained with uranyl acetate before embedding. Bar , 0.25 μm.
    Proteinase K, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 397 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    Roche dispase ii
    RPGRIP1L -knockdown in keratinocytes compromises cell-cell adhesion. ( a ) Immunofluorescence labeling of RPGRIP1L, primary cilia (ARL13B), and basal body/centriole (γ-TUB) in HaCaT cells, normal human epidermal keratinocytes (NHEK), and mouse embryonic fibroblasts (MEFs) at 48 hours after serum-starvation. Nuclei were labeled with DAPI (blue). Arrows point to centrioles or a basal body where RPGRIP1L is enriched. Bar graph represents percentage of ciliated cells (# indicates that cilium was undetectable in HaCaT cells). Scale bar, 10 μm. ( b and c ) Verification of RPGRIP1L -knockdown by qRT-PCR (b) and western blotting (c) in HaCaT cells. Values were normalized to mock transfection in (b). ( d ) Timeline of the <t>dispase</t> dissociation assay. ( e ) Dispase dissociation assay in control (Control siRNA) and RPGRIP1L -knockdown ( RPGRIP1L siRNA) HaCaT cells. ( f ) Quantification of cell fragments (mean ± SEM, n = 3 independent experiments; ** P
    Dispase Ii, supplied by Roche, used in various techniques. Bioz Stars score: 77/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    82
    Roche deciliation medium
    A–C. Thin sections of the distal tips of Tetrahymena oral (A,B) and somatic (C) cilia. The central microtubule caps (c) link the distal tips of the central microtubules to the membrane (small arrowheads) and the distal filament caps (d) link the tips of the A-tubules of each doublet to the membrane (small arrowheads). The distal filaments (see F,H,I) at the tips of somatic cilia are thin and appear identical to those seen in Chlamydomonas flagella. The more bulbous distal filaments at the tips of oral cilia appear to be unique to Tetrahymena . D. Tetrahymena cilia purified after dibucaine <t>deciliation.</t> Cilia are intact and are completely enclosed by ciliary membranes. E. Purified ciliary membrane vesicles. F. Axoneme after demembranation with 1% NP-40. Distal filament caps at the tips of A tubules (d) and the central microtubule cap (c) crowns the tip of the central microtubules. G. Distal tip of an axoneme after extraction with MgCl2 to release the capping structures. The tips of the A and central microtubules are intact but lack distal filaments and central microtubule caps (arrows). H,I. Negatively stained MgHSS containing central microtubule caps (c) and distal filaments (d) released from axonemes by MgCl2 .
    Deciliation Medium, supplied by Roche, used in various techniques. Bioz Stars score: 82/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    Roche ebc2 buffer
    A–C. Thin sections of the distal tips of Tetrahymena oral (A,B) and somatic (C) cilia. The central microtubule caps (c) link the distal tips of the central microtubules to the membrane (small arrowheads) and the distal filament caps (d) link the tips of the A-tubules of each doublet to the membrane (small arrowheads). The distal filaments (see F,H,I) at the tips of somatic cilia are thin and appear identical to those seen in Chlamydomonas flagella. The more bulbous distal filaments at the tips of oral cilia appear to be unique to Tetrahymena . D. Tetrahymena cilia purified after dibucaine <t>deciliation.</t> Cilia are intact and are completely enclosed by ciliary membranes. E. Purified ciliary membrane vesicles. F. Axoneme after demembranation with 1% NP-40. Distal filament caps at the tips of A tubules (d) and the central microtubule cap (c) crowns the tip of the central microtubules. G. Distal tip of an axoneme after extraction with MgCl2 to release the capping structures. The tips of the A and central microtubules are intact but lack distal filaments and central microtubule caps (arrows). H,I. Negatively stained MgHSS containing central microtubule caps (c) and distal filaments (d) released from axonemes by MgCl2 .
    Ebc2 Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 77/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Halo of Lewy bodies is fuzzy and contains ubiquitin, whereas the core consists of compacted α-synuclein. Transmission electron microscopic images of Lewy bodies without ( top panel ) and after proteinase K digestion ( bottom panel ). Lewy bodies were affinity-isolated with anti-α-synuclein antiserum from brain cortex and immunostained with anti-ubiquitin and anti-synuclein primary and gold particle-labeled secondary antibodies and counterstained with uranyl acetate before embedding. Bar , 0.25 μm.

    Journal:

    Article Title: Transglutaminase-mediated Intramolecular Cross-linking of Membrane-bound ?-Synuclein Promotes Amyloid Formation in Lewy Bodies

    doi: 10.1074/jbc.M109.033969

    Figure Lengend Snippet: Halo of Lewy bodies is fuzzy and contains ubiquitin, whereas the core consists of compacted α-synuclein. Transmission electron microscopic images of Lewy bodies without ( top panel ) and after proteinase K digestion ( bottom panel ). Lewy bodies were affinity-isolated with anti-α-synuclein antiserum from brain cortex and immunostained with anti-ubiquitin and anti-synuclein primary and gold particle-labeled secondary antibodies and counterstained with uranyl acetate before embedding. Bar , 0.25 μm.

    Article Snippet: Sample proteins were supplemented with ∼ 0.1 pmol of GGEL (Sigma) as internal (light) standard, subjected to limited acid hydrolysis (0.5 n trifluoroacetic acid) at 95 °C for 16 h), dried, and then digested with proteinase K (Roche Applied Science, 20 μg/ml in 10 m m HEPES-Na, pH 8.0, and 1 m m CaCl2 ) in water containing 20% H2 18 O (Aldrich) at 56 °C overnight.

    Techniques: Transmission Assay, Isolation, Labeling

    γ-Glutamyl-ϵ-lysine cross-links in brain fractions accumulate in α-synuclein immunopositive chaotrope-insoluble inclusions/Lewy bodies.  The relative abundance of GGEL was determined by enzymatic hydrolysis of sample proteins in 18 O-water and comparison with light internal GGEL standard by mass spectrometry. GGEL was related to total amino acid content.  A , GGEL accumulation associates with regions affected by degenerative changes in human brain specimen affected by Lewy body dementia ( LBD ), Alzheimer disease, or in age-matched controls ( CTRL ). Over 50% of total tissue GGEL content was recovered by either anti-ubiquitin or anti-α-synuclein affinity isolation from chaotrope-, detergent-, and thiol-insoluble brain proteins ( B ), indicating that most of the cross-linked inclusions expose both antigens in AD and LBD. Lewy bodies were exhaustively digested with proteinase K. Residual proteins remaining after digestions were dissolved in guanidine HCl and co-precipitated with a different protein (egg albumin) to disrupt β-sheet amyloid arrays between proteins. The GGEL density in proteinase K-resistant fractions is by an order of magnitude lower than in the proteinase-digestible part ( C ).  Bars  represent the means of duplicate determinations from three different donors.  Asterisks  and  hatch marks  indicate significant ( p <  0.05) differences from the corresponding controls and AD samples, respectively.

    Journal:

    Article Title: Transglutaminase-mediated Intramolecular Cross-linking of Membrane-bound ?-Synuclein Promotes Amyloid Formation in Lewy Bodies

    doi: 10.1074/jbc.M109.033969

    Figure Lengend Snippet: γ-Glutamyl-ϵ-lysine cross-links in brain fractions accumulate in α-synuclein immunopositive chaotrope-insoluble inclusions/Lewy bodies. The relative abundance of GGEL was determined by enzymatic hydrolysis of sample proteins in 18 O-water and comparison with light internal GGEL standard by mass spectrometry. GGEL was related to total amino acid content. A , GGEL accumulation associates with regions affected by degenerative changes in human brain specimen affected by Lewy body dementia ( LBD ), Alzheimer disease, or in age-matched controls ( CTRL ). Over 50% of total tissue GGEL content was recovered by either anti-ubiquitin or anti-α-synuclein affinity isolation from chaotrope-, detergent-, and thiol-insoluble brain proteins ( B ), indicating that most of the cross-linked inclusions expose both antigens in AD and LBD. Lewy bodies were exhaustively digested with proteinase K. Residual proteins remaining after digestions were dissolved in guanidine HCl and co-precipitated with a different protein (egg albumin) to disrupt β-sheet amyloid arrays between proteins. The GGEL density in proteinase K-resistant fractions is by an order of magnitude lower than in the proteinase-digestible part ( C ). Bars represent the means of duplicate determinations from three different donors. Asterisks and hatch marks indicate significant ( p < 0.05) differences from the corresponding controls and AD samples, respectively.

    Article Snippet: Sample proteins were supplemented with ∼ 0.1 pmol of GGEL (Sigma) as internal (light) standard, subjected to limited acid hydrolysis (0.5 n trifluoroacetic acid) at 95 °C for 16 h), dried, and then digested with proteinase K (Roche Applied Science, 20 μg/ml in 10 m m HEPES-Na, pH 8.0, and 1 m m CaCl2 ) in water containing 20% H2 18 O (Aldrich) at 56 °C overnight.

    Techniques: Mass Spectrometry, Isolation

    RPGRIP1L -knockdown in keratinocytes compromises cell-cell adhesion. ( a ) Immunofluorescence labeling of RPGRIP1L, primary cilia (ARL13B), and basal body/centriole (γ-TUB) in HaCaT cells, normal human epidermal keratinocytes (NHEK), and mouse embryonic fibroblasts (MEFs) at 48 hours after serum-starvation. Nuclei were labeled with DAPI (blue). Arrows point to centrioles or a basal body where RPGRIP1L is enriched. Bar graph represents percentage of ciliated cells (# indicates that cilium was undetectable in HaCaT cells). Scale bar, 10 μm. ( b and c ) Verification of RPGRIP1L -knockdown by qRT-PCR (b) and western blotting (c) in HaCaT cells. Values were normalized to mock transfection in (b). ( d ) Timeline of the dispase dissociation assay. ( e ) Dispase dissociation assay in control (Control siRNA) and RPGRIP1L -knockdown ( RPGRIP1L siRNA) HaCaT cells. ( f ) Quantification of cell fragments (mean ± SEM, n = 3 independent experiments; ** P

    Journal: PLoS Genetics

    Article Title: RPGRIP1L is required for stabilizing epidermal keratinocyte adhesion through regulating desmoglein endocytosis

    doi: 10.1371/journal.pgen.1007914

    Figure Lengend Snippet: RPGRIP1L -knockdown in keratinocytes compromises cell-cell adhesion. ( a ) Immunofluorescence labeling of RPGRIP1L, primary cilia (ARL13B), and basal body/centriole (γ-TUB) in HaCaT cells, normal human epidermal keratinocytes (NHEK), and mouse embryonic fibroblasts (MEFs) at 48 hours after serum-starvation. Nuclei were labeled with DAPI (blue). Arrows point to centrioles or a basal body where RPGRIP1L is enriched. Bar graph represents percentage of ciliated cells (# indicates that cilium was undetectable in HaCaT cells). Scale bar, 10 μm. ( b and c ) Verification of RPGRIP1L -knockdown by qRT-PCR (b) and western blotting (c) in HaCaT cells. Values were normalized to mock transfection in (b). ( d ) Timeline of the dispase dissociation assay. ( e ) Dispase dissociation assay in control (Control siRNA) and RPGRIP1L -knockdown ( RPGRIP1L siRNA) HaCaT cells. ( f ) Quantification of cell fragments (mean ± SEM, n = 3 independent experiments; ** P

    Article Snippet: Subsequently, cells were washed with DPBS and incubated with dispase II (2.4 U/ml in EMEM with 10% FBS and 1.5 mM CaCl2 , Roche, Indianapolis, IN), for 20 min at 37°C.

    Techniques: Immunofluorescence, Labeling, Quantitative RT-PCR, Western Blot, Transfection

    A–C. Thin sections of the distal tips of Tetrahymena oral (A,B) and somatic (C) cilia. The central microtubule caps (c) link the distal tips of the central microtubules to the membrane (small arrowheads) and the distal filament caps (d) link the tips of the A-tubules of each doublet to the membrane (small arrowheads). The distal filaments (see F,H,I) at the tips of somatic cilia are thin and appear identical to those seen in Chlamydomonas flagella. The more bulbous distal filaments at the tips of oral cilia appear to be unique to Tetrahymena . D. Tetrahymena cilia purified after dibucaine deciliation. Cilia are intact and are completely enclosed by ciliary membranes. E. Purified ciliary membrane vesicles. F. Axoneme after demembranation with 1% NP-40. Distal filament caps at the tips of A tubules (d) and the central microtubule cap (c) crowns the tip of the central microtubules. G. Distal tip of an axoneme after extraction with MgCl2 to release the capping structures. The tips of the A and central microtubules are intact but lack distal filaments and central microtubule caps (arrows). H,I. Negatively stained MgHSS containing central microtubule caps (c) and distal filaments (d) released from axonemes by MgCl2 .

    Journal:

    Article Title: Discovery and functional evaluation of ciliary proteins in Tetrahymena thermophila

    doi: 10.1016/B978-0-12-397944-5.00013-4

    Figure Lengend Snippet: A–C. Thin sections of the distal tips of Tetrahymena oral (A,B) and somatic (C) cilia. The central microtubule caps (c) link the distal tips of the central microtubules to the membrane (small arrowheads) and the distal filament caps (d) link the tips of the A-tubules of each doublet to the membrane (small arrowheads). The distal filaments (see F,H,I) at the tips of somatic cilia are thin and appear identical to those seen in Chlamydomonas flagella. The more bulbous distal filaments at the tips of oral cilia appear to be unique to Tetrahymena . D. Tetrahymena cilia purified after dibucaine deciliation. Cilia are intact and are completely enclosed by ciliary membranes. E. Purified ciliary membrane vesicles. F. Axoneme after demembranation with 1% NP-40. Distal filament caps at the tips of A tubules (d) and the central microtubule cap (c) crowns the tip of the central microtubules. G. Distal tip of an axoneme after extraction with MgCl2 to release the capping structures. The tips of the A and central microtubules are intact but lack distal filaments and central microtubule caps (arrows). H,I. Negatively stained MgHSS containing central microtubule caps (c) and distal filaments (d) released from axonemes by MgCl2 .

    Article Snippet: Collect cells by centrifugation (1700 × g 3 min; swinging bucket rotor, 50 ml conical tubes), wash once with 10 mM Tris-HCl pH 7.5 and gently suspend in 20 ml of the deciliation medium (10 mM Tris-HCl pH 7.4, 50 mM sucrose, 10 mM CaCl2 , protease inhibitors (Complete, Roche)) in a 250 ml flask.

    Techniques: Purification, Staining