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Promega cacl2
Cacl2, supplied by Promega, used in various techniques. Bioz Stars score: 96/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 96 stars, based on 5 article reviews
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cacl2 - by Bioz Stars, 2020-02
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Centrifugation:

Article Title: Chemical Proteomics Identifies Druggable Vulnerabilities in a Genetically Defined Cancer
Article Snippet: The bead mixture was diluted with 900 μL PBS, pelleted by centrifugation (1,400 × g, 3 min), and resuspended in PBS containing 2 M urea (200 μL). .. To this was added 1 mM CaCl2 (2 μL of a 200 mM stock in water) and trypsin (2 μg, Promega, sequencing grade) and the digestion was allowed to proceed overnight at 37 °C with shaking.

Article Title: An allosteric propofol-binding site in kinesin disrupts kinesin-mediated processive movement on microtubules
Article Snippet: Subsequently, 1 μl of 0.1 m CaCl2 and 1 μl of 1% ProteaseMaxTM Surfactant Enhancer (w/v%) was added, and proteins were digested overnight at 37 °C using sequencing grade trypsin (Promega) at ∼1:20 protease:protein (w/w) ratio. .. Insoluble debris was removed by centrifugation at 16,000 × g and the soluble peptide digest was desalted using C18 stage tips prepared in-house.

Article Title: Coordinated responses to individual tumor antigens by IgG antibody and CD8+ T cells following cancer vaccination
Article Snippet: In brief, samples were dried, dissolved in 10 μL of 4X buffer (8 M urea,1 M Tris (pH 8.5), 8 mM CaCl2 , 0.2 M methylamine), reduced, alkylated, diluted to a final 2 M urea concentration and digested by addition of 1.6 μg of sequencing grade trypsin overnight (ProMega) Completion of the digestion was confirmed by 1-D gel analysis. .. Twenty-five μg of each digested sample was then solid phase extracted using Oasis HLB 1cm3 cartridges (Waters Corporation), and peptides dried by vacuum centrifugation.

Article Title: Raf Kinase Inhibitor Protein (RKIP) Blocks Signal Transducer and Activator of Transcription 3 (STAT3) Activation in Breast and Prostate Cancer
Article Snippet: After rehydratation for 15 minutes at 4°C in digestion buffer consisting of 0.05 M ammonium bicarbonate, 5 mM CaCl2 and 12.5 μg/mL trypsin (porcine, sequence grade, Promega, Madison, WI, USA), the proteins were digested. .. After digestion, the peptides were recovered from the mixture by centrifugation (Eppendorf centrifuge).

Luciferase:

Article Title: Calcium Depletion Dissociates and Activates Heterodimeric Notch Receptors
Article Snippet: .. At 24 h after transfection, the cells were briefly treated with HBS containing various concentrations of EDTA or 2.5 mM CaCl2 as described above and then allowed to recover in D10 for up to 8 h. Firefly and Renilla luciferase activities were determined in whole cell extracts using the Dual Luciferase Assay Kit (Promega) and a Turner Designs TD20 dual luminometer. .. Luciferase activity assays were also conducted with HeLa cells using a procedure modified from that described by Jarriault et al. ( ).

Article Title: Spatial specificity of auxin responses coordinates wood formation
Article Snippet: The transfected protoplasts were diluted with 240 mM CaCl2 (1:3) followed by cell lysis and dual-luciferase assay using the Dual-Luciferase Reporter Assay System (Promega) and following the manufacturer’s instructions. .. For experimental analysis, Firefly Luciferase activity was normalized to Renilla Luciferase activity.

Reporter Assay:

Article Title: Spatial specificity of auxin responses coordinates wood formation
Article Snippet: .. The transfected protoplasts were diluted with 240 mM CaCl2 (1:3) followed by cell lysis and dual-luciferase assay using the Dual-Luciferase Reporter Assay System (Promega) and following the manufacturer’s instructions. .. Luminescence was measured using a Synergy H4 Hybrid Multiplate Reader (BioTek).

Autoradiography:

Article Title: Both Ser361 phosphorylation and the C‐arrestin domain of thioredoxin interacting protein are important for cell cycle blockade at the G1/S checkpoint
Article Snippet: For the PKC reaction, 20 mm Tris/HCl pH 7.5, 5 mm MgCl2 , 1 mm CaCl2 , 320 ng·μL−1 phosphatidylserine, 32 ng·μL−1 diacylglycerol, and 3 ng·μL−1 PKC (Promega) were added to the kinase reaction mix. .. The gel was dried and radioactive phosphorylated proteins were detected by autoradiography.

Article Title: Assembly and Function of the RNA Editing Complex in Trypanosoma brucei Requires Band III Protein
Article Snippet: U deletion reactions were in 10 mM KCl-MRB (25 mM Tris-HCl [pH 7.5], 10 mM magnesium acetate, 10 mM KCl, 1mM EDTA, 0.5 mM dithiothreitol [DTT], 5% glycerol) supplemented with 0.15 mM ATP, 3 mM ADP, 5 mM CaCl2 , 4.5 ng of torula RNA per μl, 5 mM DTT, 0.8 U of RNasin per ml (Promega). .. The 20-μl reaction mixtures were incubated for 40 min at 22°C, and recovered RNAs were resolved on 9% polyacrylamide-7.5 M urea gels and visualized by autoradiography.

Construct:

Article Title: Spatial specificity of auxin responses coordinates wood formation
Article Snippet: For transfection, we used 10 µg of reporter construct ( pKB55 ) containing p35S:LUC ( Renilla ) as an internal control and 10 µg of each effector construct. .. The transfected protoplasts were diluted with 240 mM CaCl2 (1:3) followed by cell lysis and dual-luciferase assay using the Dual-Luciferase Reporter Assay System (Promega) and following the manufacturer’s instructions.

Incubation:

Article Title: Chemical Proteomics Identifies Druggable Vulnerabilities in a Genetically Defined Cancer
Article Snippet: To this was added 10 mM DTT (25 μL of a 200 mM stock in water) and the beads were incubated at 65 °C for 15 mins. .. To this was added 1 mM CaCl2 (2 μL of a 200 mM stock in water) and trypsin (2 μg, Promega, sequencing grade) and the digestion was allowed to proceed overnight at 37 °C with shaking.

Article Title: An allosteric propofol-binding site in kinesin disrupts kinesin-mediated processive movement on microtubules
Article Snippet: Protein was then alkylated with the addition of 2.7 μl of 0.55 m iodoacetamide and incubation in the dark at room temperature for 20 min. .. Subsequently, 1 μl of 0.1 m CaCl2 and 1 μl of 1% ProteaseMaxTM Surfactant Enhancer (w/v%) was added, and proteins were digested overnight at 37 °C using sequencing grade trypsin (Promega) at ∼1:20 protease:protein (w/w) ratio.

Article Title: Novel Citronellyl-Based Photoprobes Designed to Identify ER Proteins Interacting with Dolichyl Phosphate in Yeast and Mammalian Cells
Article Snippet: Biotinylated proteins were captured and recovered by incubation with streptavidin beads (Thermo Scientific, 0.1 mL, 50% slurry) in PBS/0.2 % SDS for 2 hours with rocking. .. After washing with PBS, the beads were resuspended in 200 μl 2M urea/PBS supplemented with CaCl2 (1 mM) for on-bead digestion with trypsin (Promega, 2 μg per 0.2 mL reaction) at 37°C overnight.

Article Title: Both Ser361 phosphorylation and the C‐arrestin domain of thioredoxin interacting protein are important for cell cycle blockade at the G1/S checkpoint
Article Snippet: In vitro kinase assay One milligram of purified FLAG–TXNIP was incubated at 30 °C for 10 min in kinase reaction mix (2.96 MBq·mL−1 γ‐[32 P]ATP, 2 μg·mL−1 200 μm ATP, and 0.2 mm dithiothreitol). .. For the PKC reaction, 20 mm Tris/HCl pH 7.5, 5 mm MgCl2 , 1 mm CaCl2 , 320 ng·μL−1 phosphatidylserine, 32 ng·μL−1 diacylglycerol, and 3 ng·μL−1 PKC (Promega) were added to the kinase reaction mix.

Article Title: SIRT4 is a Lysine Deacylase that Controls Leucine Metabolism and Insulin Secretion
Article Snippet: The agarose beads were subsequently eluted again by incubation with 150 μl of 8M-containing buffer (8 M urea in 50 mM Tris, pH 8.0, 40 mM NaCl, 2 mM MgCl2 , + 1× Complete Roche protease inhibitor tablet, 10 mM Nicotinamide, 10 μM TSA) for 15 minutes at room temperature while rotating. .. Following dilution to 1.5 M urea with 50 mM Tris (pH 8.0), 5 mM CaCl2 , 5 μg of sequencing-grade trypsin (Promega) was added and digestion proceeded overnight at 37°C.

Article Title: Identification of Cisplatin-Binding Proteins Using Agarose Conjugates of Platinum Compounds
Article Snippet: The samples were reduced in 10 mM DTT/100 mM ammonium bicarbonate, alkylated in 55 mM iodoacetamide/100 mM ammonium bicarbonate, and dehydrated gels re-swelled in digestion buffer containing 50 mM ammonium bicarbonate/5 mM CaCl2 /12.5 ng/ µl sequencing grade modified trypsin (Promega, Madison, WI). .. Following an overnight incubation at 37°C, extracted peptides were identified by collection of MS/MS data using a Velos Pro linear ion trap mass spectrometer (ThermoFisher, San Jose, CA).

Article Title: Assembly and Function of the RNA Editing Complex in Trypanosoma brucei Requires Band III Protein
Article Snippet: U deletion reactions were in 10 mM KCl-MRB (25 mM Tris-HCl [pH 7.5], 10 mM magnesium acetate, 10 mM KCl, 1mM EDTA, 0.5 mM dithiothreitol [DTT], 5% glycerol) supplemented with 0.15 mM ATP, 3 mM ADP, 5 mM CaCl2 , 4.5 ng of torula RNA per μl, 5 mM DTT, 0.8 U of RNasin per ml (Promega). .. The 20-μl reaction mixtures were incubated for 40 min at 22°C, and recovered RNAs were resolved on 9% polyacrylamide-7.5 M urea gels and visualized by autoradiography.

Stripping Membranes:

Article Title: SIRT4 is a Lysine Deacylase that Controls Leucine Metabolism and Insulin Secretion
Article Snippet: After the pH was verified to be ~7–8 with a pH strip, the sample was reduced with 5 mM DTT at 37°C for 30 min, cooled to RT, alkylate d with 15 mM iodoacetamide for 30 min in the dark, and unreacted iodoacetamide quenched by the addition of DTT up to 15 mM for 10 min at room temperature. .. Following dilution to 1.5 M urea with 50 mM Tris (pH 8.0), 5 mM CaCl2 , 5 μg of sequencing-grade trypsin (Promega) was added and digestion proceeded overnight at 37°C.

Activity Assay:

Article Title: Calcium Depletion Dissociates and Activates Heterodimeric Notch Receptors
Article Snippet: At 24 h after transfection, the cells were briefly treated with HBS containing various concentrations of EDTA or 2.5 mM CaCl2 as described above and then allowed to recover in D10 for up to 8 h. Firefly and Renilla luciferase activities were determined in whole cell extracts using the Dual Luciferase Assay Kit (Promega) and a Turner Designs TD20 dual luminometer. .. Luciferase activity assays were also conducted with HeLa cells using a procedure modified from that described by Jarriault et al. ( ).

Article Title: Spatial specificity of auxin responses coordinates wood formation
Article Snippet: Paragraph title: Transient reporter activity assays ... The transfected protoplasts were diluted with 240 mM CaCl2 (1:3) followed by cell lysis and dual-luciferase assay using the Dual-Luciferase Reporter Assay System (Promega) and following the manufacturer’s instructions.

Mass Spectrometry:

Article Title: Control of seed dormancy and germination by DOG1-AHG1 PP2C phosphatase complex via binding to heme
Article Snippet: Paragraph title: Mass spectrometry and data analysis ... Proteins were digested for 18 h at 37 °C in 2 M urea, 100 mM Tris pH 8.5, 1 mM CaCl2 with 2 μg trypsin (Promega).

Article Title: An allosteric propofol-binding site in kinesin disrupts kinesin-mediated processive movement on microtubules
Article Snippet: Subsequently, 1 μl of 0.1 m CaCl2 and 1 μl of 1% ProteaseMaxTM Surfactant Enhancer (w/v%) was added, and proteins were digested overnight at 37 °C using sequencing grade trypsin (Promega) at ∼1:20 protease:protein (w/w) ratio. .. The final elution was dried by SpeedVac and prior to MS analysis was resuspended in 0.1% formic acid (v/v %).

Article Title: Novel Citronellyl-Based Photoprobes Designed to Identify ER Proteins Interacting with Dolichyl Phosphate in Yeast and Mammalian Cells
Article Snippet: After washing with PBS, the beads were resuspended in 200 μl 2M urea/PBS supplemented with CaCl2 (1 mM) for on-bead digestion with trypsin (Promega, 2 μg per 0.2 mL reaction) at 37°C overnight. .. Tryptic peptides were loaded onto the cartridges in 5% acetonitrile/0.1% TFA, washed with 25 mL 5% acetonitrile/0.1% TFA, eluted with 0.6 mL 70% acetonitrile/0.1% TFA and 0.4 mL 90% acetonitrile/0.1% TFA, dried under N2 and stored at −80°C until analysis by mass spectrometry as described below.

Article Title: Targeted Identification of Glycosylated Proteins in the Gastric Pathogen Helicobacter pylori (Hp) *
Article Snippet: The samples were diluted by a factor of four with 100 mm Tris-HCl (pH 8.5) and 1 mm CaCl2 , and then digested with 0.25 μg of trypsin (Promega) overnight at 37 °C. .. The Vincent J. Coates Proteomics/Mass Spectrometry Laboratory at University of California Berkeley performed mass spectrometry analyses of the enriched, azide-labeled sample and mock-enriched control sample.

Article Title: Coordinated responses to individual tumor antigens by IgG antibody and CD8+ T cells following cancer vaccination
Article Snippet: TMT LC-MS/MS of 4T1 cells and autophagosome-enriched vaccine Quantitative tandem mass tag (TMT) liquid chromatography tandem mass spectrometry (LC-MS/MS) was performed by the Proteomics Shared Resource at Oregon Health & Science University on three 4T1 autophagosome-enriched vaccine lots and three paired samples of untreated whole 4T1 cells. .. In brief, samples were dried, dissolved in 10 μL of 4X buffer (8 M urea,1 M Tris (pH 8.5), 8 mM CaCl2 , 0.2 M methylamine), reduced, alkylated, diluted to a final 2 M urea concentration and digested by addition of 1.6 μg of sequencing grade trypsin overnight (ProMega) Completion of the digestion was confirmed by 1-D gel analysis.

Article Title: Identification of Cisplatin-Binding Proteins Using Agarose Conjugates of Platinum Compounds
Article Snippet: Paragraph title: Mass spectrometry ... The samples were reduced in 10 mM DTT/100 mM ammonium bicarbonate, alkylated in 55 mM iodoacetamide/100 mM ammonium bicarbonate, and dehydrated gels re-swelled in digestion buffer containing 50 mM ammonium bicarbonate/5 mM CaCl2 /12.5 ng/ µl sequencing grade modified trypsin (Promega, Madison, WI).

BIA-KA:

Article Title: Coordinated responses to individual tumor antigens by IgG antibody and CD8+ T cells following cancer vaccination
Article Snippet: Samples were lysed using a probe sonicator and protein concentration was estimated using BCA assay. .. In brief, samples were dried, dissolved in 10 μL of 4X buffer (8 M urea,1 M Tris (pH 8.5), 8 mM CaCl2 , 0.2 M methylamine), reduced, alkylated, diluted to a final 2 M urea concentration and digested by addition of 1.6 μg of sequencing grade trypsin overnight (ProMega) Completion of the digestion was confirmed by 1-D gel analysis.

Modification:

Article Title: Calcium Depletion Dissociates and Activates Heterodimeric Notch Receptors
Article Snippet: At 24 h after transfection, the cells were briefly treated with HBS containing various concentrations of EDTA or 2.5 mM CaCl2 as described above and then allowed to recover in D10 for up to 8 h. Firefly and Renilla luciferase activities were determined in whole cell extracts using the Dual Luciferase Assay Kit (Promega) and a Turner Designs TD20 dual luminometer. .. Luciferase activity assays were also conducted with HeLa cells using a procedure modified from that described by Jarriault et al. ( ).

Article Title: The WHHERE coactivator complex is required for retinoic acid-dependent regulation of embryonic symmetry
Article Snippet: .. Urea was then diluted to 2 M with 0.1 M Tris-HCl pH 8.5, CaCl2 was added to 0.5 mM and modified trypsin (Promega), 1:100 (wt/wt), was added for over 12 h at 37 °C. ..

Article Title: Human Family with Sequence Similarity 60 Member A (FAM60A) Protein: a New Subunit of the Sin3 Deacetylase Complex *
Article Snippet: .. Urea was then diluted to 2 m with 0.1 m Tris-HCl, pH 8.5, CaCl2 was added to 0.5 m m , and modified trypsin (Promega), 1:100 (wt/wt), was added for over 12 h at 37 °C. ..

Article Title: Identification of Cisplatin-Binding Proteins Using Agarose Conjugates of Platinum Compounds
Article Snippet: .. The samples were reduced in 10 mM DTT/100 mM ammonium bicarbonate, alkylated in 55 mM iodoacetamide/100 mM ammonium bicarbonate, and dehydrated gels re-swelled in digestion buffer containing 50 mM ammonium bicarbonate/5 mM CaCl2 /12.5 ng/ µl sequencing grade modified trypsin (Promega, Madison, WI). .. Following an overnight incubation at 37°C, extracted peptides were identified by collection of MS/MS data using a Velos Pro linear ion trap mass spectrometer (ThermoFisher, San Jose, CA).

Kinase Assay:

Article Title: Both Ser361 phosphorylation and the C‐arrestin domain of thioredoxin interacting protein are important for cell cycle blockade at the G1/S checkpoint
Article Snippet: Paragraph title: In vitro kinase assay ... For the PKC reaction, 20 mm Tris/HCl pH 7.5, 5 mm MgCl2 , 1 mm CaCl2 , 320 ng·μL−1 phosphatidylserine, 32 ng·μL−1 diacylglycerol, and 3 ng·μL−1 PKC (Promega) were added to the kinase reaction mix.

Derivative Assay:

Article Title: Spatial specificity of auxin responses coordinates wood formation
Article Snippet: For transient reporter activity assays, protoplasts derived from an Arabidopsis (Col-0) dark-grown root cell suspension culture (kindly provided by Claudia Jonak, GMI, Vienna) were isolated and transfected. .. The transfected protoplasts were diluted with 240 mM CaCl2 (1:3) followed by cell lysis and dual-luciferase assay using the Dual-Luciferase Reporter Assay System (Promega) and following the manufacturer’s instructions.

Transfection:

Article Title: Calcium Depletion Dissociates and Activates Heterodimeric Notch Receptors
Article Snippet: .. At 24 h after transfection, the cells were briefly treated with HBS containing various concentrations of EDTA or 2.5 mM CaCl2 as described above and then allowed to recover in D10 for up to 8 h. Firefly and Renilla luciferase activities were determined in whole cell extracts using the Dual Luciferase Assay Kit (Promega) and a Turner Designs TD20 dual luminometer. .. Luciferase activity assays were also conducted with HeLa cells using a procedure modified from that described by Jarriault et al. ( ).

Article Title: Spatial specificity of auxin responses coordinates wood formation
Article Snippet: .. The transfected protoplasts were diluted with 240 mM CaCl2 (1:3) followed by cell lysis and dual-luciferase assay using the Dual-Luciferase Reporter Assay System (Promega) and following the manufacturer’s instructions. .. Luminescence was measured using a Synergy H4 Hybrid Multiplate Reader (BioTek).

Article Title: Raf Kinase Inhibitor Protein (RKIP) Blocks Signal Transducer and Activator of Transcription 3 (STAT3) Activation in Breast and Prostate Cancer
Article Snippet: Identification of Proteins Interacting with RKIP via Liquid Chromatography-Mass Spectrometry (LC-MS/MS) Cells were transfected with HA-RKIP for 48 h. Lysed cells were immunoprecipitated with an antibody to HA and proteins were separated via 10% SDS-PAGE. .. After rehydratation for 15 minutes at 4°C in digestion buffer consisting of 0.05 M ammonium bicarbonate, 5 mM CaCl2 and 12.5 μg/mL trypsin (porcine, sequence grade, Promega, Madison, WI, USA), the proteins were digested.

Chromatography:

Article Title: Raf Kinase Inhibitor Protein (RKIP) Blocks Signal Transducer and Activator of Transcription 3 (STAT3) Activation in Breast and Prostate Cancer
Article Snippet: Paragraph title: Identification of Proteins Interacting with RKIP via Liquid Chromatography-Mass Spectrometry (LC-MS/MS) ... After rehydratation for 15 minutes at 4°C in digestion buffer consisting of 0.05 M ammonium bicarbonate, 5 mM CaCl2 and 12.5 μg/mL trypsin (porcine, sequence grade, Promega, Madison, WI, USA), the proteins were digested.

Immunoprecipitation:

Article Title: Raf Kinase Inhibitor Protein (RKIP) Blocks Signal Transducer and Activator of Transcription 3 (STAT3) Activation in Breast and Prostate Cancer
Article Snippet: Identification of Proteins Interacting with RKIP via Liquid Chromatography-Mass Spectrometry (LC-MS/MS) Cells were transfected with HA-RKIP for 48 h. Lysed cells were immunoprecipitated with an antibody to HA and proteins were separated via 10% SDS-PAGE. .. After rehydratation for 15 minutes at 4°C in digestion buffer consisting of 0.05 M ammonium bicarbonate, 5 mM CaCl2 and 12.5 μg/mL trypsin (porcine, sequence grade, Promega, Madison, WI, USA), the proteins were digested.

Protease Inhibitor:

Article Title: SIRT4 is a Lysine Deacylase that Controls Leucine Metabolism and Insulin Secretion
Article Snippet: The agarose beads were subsequently eluted again by incubation with 150 μl of 8M-containing buffer (8 M urea in 50 mM Tris, pH 8.0, 40 mM NaCl, 2 mM MgCl2 , + 1× Complete Roche protease inhibitor tablet, 10 mM Nicotinamide, 10 μM TSA) for 15 minutes at room temperature while rotating. .. Following dilution to 1.5 M urea with 50 mM Tris (pH 8.0), 5 mM CaCl2 , 5 μg of sequencing-grade trypsin (Promega) was added and digestion proceeded overnight at 37°C.

Liquid Chromatography:

Article Title: Coordinated responses to individual tumor antigens by IgG antibody and CD8+ T cells following cancer vaccination
Article Snippet: TMT LC-MS/MS of 4T1 cells and autophagosome-enriched vaccine Quantitative tandem mass tag (TMT) liquid chromatography tandem mass spectrometry (LC-MS/MS) was performed by the Proteomics Shared Resource at Oregon Health & Science University on three 4T1 autophagosome-enriched vaccine lots and three paired samples of untreated whole 4T1 cells. .. In brief, samples were dried, dissolved in 10 μL of 4X buffer (8 M urea,1 M Tris (pH 8.5), 8 mM CaCl2 , 0.2 M methylamine), reduced, alkylated, diluted to a final 2 M urea concentration and digested by addition of 1.6 μg of sequencing grade trypsin overnight (ProMega) Completion of the digestion was confirmed by 1-D gel analysis.

Protein Concentration:

Article Title: Coordinated responses to individual tumor antigens by IgG antibody and CD8+ T cells following cancer vaccination
Article Snippet: Samples were lysed using a probe sonicator and protein concentration was estimated using BCA assay. .. In brief, samples were dried, dissolved in 10 μL of 4X buffer (8 M urea,1 M Tris (pH 8.5), 8 mM CaCl2 , 0.2 M methylamine), reduced, alkylated, diluted to a final 2 M urea concentration and digested by addition of 1.6 μg of sequencing grade trypsin overnight (ProMega) Completion of the digestion was confirmed by 1-D gel analysis.

Sequencing:

Article Title: Chemical Proteomics Identifies Druggable Vulnerabilities in a Genetically Defined Cancer
Article Snippet: .. To this was added 1 mM CaCl2 (2 μL of a 200 mM stock in water) and trypsin (2 μg, Promega, sequencing grade) and the digestion was allowed to proceed overnight at 37 °C with shaking. .. The beads were separated from the digest with Micro Bio-Spin columns (Bio-Rad) by centrifugation (1,000 × g, 1 min), washed (2 × 1 mL PBS and 2 × 1 mL water) and then transferred to fresh eppendorf tubes with 1 mL water.

Article Title: An allosteric propofol-binding site in kinesin disrupts kinesin-mediated processive movement on microtubules
Article Snippet: .. Subsequently, 1 μl of 0.1 m CaCl2 and 1 μl of 1% ProteaseMaxTM Surfactant Enhancer (w/v%) was added, and proteins were digested overnight at 37 °C using sequencing grade trypsin (Promega) at ∼1:20 protease:protein (w/w) ratio. .. To stop digestion, TFA was added to 0.5% (v/v%) and samples were incubated at room temperature for 10 min.

Article Title: Coordinated responses to individual tumor antigens by IgG antibody and CD8+ T cells following cancer vaccination
Article Snippet: .. In brief, samples were dried, dissolved in 10 μL of 4X buffer (8 M urea,1 M Tris (pH 8.5), 8 mM CaCl2 , 0.2 M methylamine), reduced, alkylated, diluted to a final 2 M urea concentration and digested by addition of 1.6 μg of sequencing grade trypsin overnight (ProMega) Completion of the digestion was confirmed by 1-D gel analysis. .. Twenty-five μg of each digested sample was then solid phase extracted using Oasis HLB 1cm3 cartridges (Waters Corporation), and peptides dried by vacuum centrifugation.

Article Title: SIRT4 is a Lysine Deacylase that Controls Leucine Metabolism and Insulin Secretion
Article Snippet: .. Following dilution to 1.5 M urea with 50 mM Tris (pH 8.0), 5 mM CaCl2 , 5 μg of sequencing-grade trypsin (Promega) was added and digestion proceeded overnight at 37°C. .. The samples were acidified to 0.5% TFA and desalted on a Waters 50 mg tC18 SEP-PAK SPE column (Waters, #WAT054960) —eluting once with 500 μl 25% acetonitrile/0.1% TFA and twice with 500 μl 50% acetonitrile/0.1% TFA.

Article Title: Raf Kinase Inhibitor Protein (RKIP) Blocks Signal Transducer and Activator of Transcription 3 (STAT3) Activation in Breast and Prostate Cancer
Article Snippet: .. After rehydratation for 15 minutes at 4°C in digestion buffer consisting of 0.05 M ammonium bicarbonate, 5 mM CaCl2 and 12.5 μg/mL trypsin (porcine, sequence grade, Promega, Madison, WI, USA), the proteins were digested. ..

Article Title: Identification of Cisplatin-Binding Proteins Using Agarose Conjugates of Platinum Compounds
Article Snippet: .. The samples were reduced in 10 mM DTT/100 mM ammonium bicarbonate, alkylated in 55 mM iodoacetamide/100 mM ammonium bicarbonate, and dehydrated gels re-swelled in digestion buffer containing 50 mM ammonium bicarbonate/5 mM CaCl2 /12.5 ng/ µl sequencing grade modified trypsin (Promega, Madison, WI). .. Following an overnight incubation at 37°C, extracted peptides were identified by collection of MS/MS data using a Velos Pro linear ion trap mass spectrometer (ThermoFisher, San Jose, CA).

Sonication:

Article Title: Novel Citronellyl-Based Photoprobes Designed to Identify ER Proteins Interacting with Dolichyl Phosphate in Yeast and Mammalian Cells
Article Snippet: After the final wash, protein pellets were resuspended in 1 mL of 1.2% SDS in PBS, heated to 100°C and sonicated briefly to dissolve completely. .. After washing with PBS, the beads were resuspended in 200 μl 2M urea/PBS supplemented with CaCl2 (1 mM) for on-bead digestion with trypsin (Promega, 2 μg per 0.2 mL reaction) at 37°C overnight.

Isolation:

Article Title: Spatial specificity of auxin responses coordinates wood formation
Article Snippet: For transient reporter activity assays, protoplasts derived from an Arabidopsis (Col-0) dark-grown root cell suspension culture (kindly provided by Claudia Jonak, GMI, Vienna) were isolated and transfected. .. The transfected protoplasts were diluted with 240 mM CaCl2 (1:3) followed by cell lysis and dual-luciferase assay using the Dual-Luciferase Reporter Assay System (Promega) and following the manufacturer’s instructions.

Protein Quantitation:

Article Title: Control of seed dormancy and germination by DOG1-AHG1 PP2C phosphatase complex via binding to heme
Article Snippet: Proteins were digested for 18 h at 37 °C in 2 M urea, 100 mM Tris pH 8.5, 1 mM CaCl2 with 2 μg trypsin (Promega). .. Protein and peptide identification and protein quantitation were done with Integrated Proteomics Pipeline—IP2 (Integrated Proteomics Applications, Inc., San Diego, CA. http://www.integratedproteomics.com/ ).

Tandem Mass Spectroscopy:

Article Title: Identification of Cisplatin-Binding Proteins Using Agarose Conjugates of Platinum Compounds
Article Snippet: The samples were reduced in 10 mM DTT/100 mM ammonium bicarbonate, alkylated in 55 mM iodoacetamide/100 mM ammonium bicarbonate, and dehydrated gels re-swelled in digestion buffer containing 50 mM ammonium bicarbonate/5 mM CaCl2 /12.5 ng/ µl sequencing grade modified trypsin (Promega, Madison, WI). .. Following an overnight incubation at 37°C, extracted peptides were identified by collection of MS/MS data using a Velos Pro linear ion trap mass spectrometer (ThermoFisher, San Jose, CA).

Labeling:

Article Title: An allosteric propofol-binding site in kinesin disrupts kinesin-mediated processive movement on microtubules
Article Snippet: Paragraph title: Photoaffinity labeling for microsequencing ... Subsequently, 1 μl of 0.1 m CaCl2 and 1 μl of 1% ProteaseMaxTM Surfactant Enhancer (w/v%) was added, and proteins were digested overnight at 37 °C using sequencing grade trypsin (Promega) at ∼1:20 protease:protein (w/w) ratio.

Article Title: Targeted Identification of Glycosylated Proteins in the Gastric Pathogen Helicobacter pylori (Hp) *
Article Snippet: Protein Identification by MudPIT An azide-labeled enriched glycoprotein sample containing 150 μg of protein and a mock enriched control (which was labeled with Ac4 GlcNAc, reacted with Phos-FLAG-His6 , and enriched via anti-FLAG and Ni-NTA affinity chromatography, as described above) were concentrated using a Microcon centrifugal filter device (Millipore) to ∼10 μl, suspended in 8 m urea and 100 mm Tris-HCl (pH 8.5), reduced with 5 mm tris (2-carboxyethyl)phosphine (TCEP), and reacted with 10 mm iodoacetamide. .. The samples were diluted by a factor of four with 100 mm Tris-HCl (pH 8.5) and 1 mm CaCl2 , and then digested with 0.25 μg of trypsin (Promega) overnight at 37 °C.

Article Title: Coordinated responses to individual tumor antigens by IgG antibody and CD8+ T cells following cancer vaccination
Article Snippet: In brief, samples were dried, dissolved in 10 μL of 4X buffer (8 M urea,1 M Tris (pH 8.5), 8 mM CaCl2 , 0.2 M methylamine), reduced, alkylated, diluted to a final 2 M urea concentration and digested by addition of 1.6 μg of sequencing grade trypsin overnight (ProMega) Completion of the digestion was confirmed by 1-D gel analysis. .. Samples were labeled with 10-plex TMT reagents (Thermo Scientific), pooled together, and on-line two dimensional reverse phase/reverse phase (RP-RP) liquid chromatography used to separate into 9 fractions at high pH, and each fraction further separated at low pH.

Purification:

Article Title: Control of seed dormancy and germination by DOG1-AHG1 PP2C phosphatase complex via binding to heme
Article Snippet: Mass spectrometry and data analysis Purified proteins were reduced with 5 mM Tris (2-carboxyethyl) phosphine hydrochloride (Sigma-Aldrich) and alkylated. .. Proteins were digested for 18 h at 37 °C in 2 M urea, 100 mM Tris pH 8.5, 1 mM CaCl2 with 2 μg trypsin (Promega).

Article Title: Both Ser361 phosphorylation and the C‐arrestin domain of thioredoxin interacting protein are important for cell cycle blockade at the G1/S checkpoint
Article Snippet: In vitro kinase assay One milligram of purified FLAG–TXNIP was incubated at 30 °C for 10 min in kinase reaction mix (2.96 MBq·mL−1 γ‐[32 P]ATP, 2 μg·mL−1 200 μm ATP, and 0.2 mm dithiothreitol). .. For the PKC reaction, 20 mm Tris/HCl pH 7.5, 5 mm MgCl2 , 1 mm CaCl2 , 320 ng·μL−1 phosphatidylserine, 32 ng·μL−1 diacylglycerol, and 3 ng·μL−1 PKC (Promega) were added to the kinase reaction mix.

Affinity Chromatography:

Article Title: Targeted Identification of Glycosylated Proteins in the Gastric Pathogen Helicobacter pylori (Hp) *
Article Snippet: Protein Identification by MudPIT An azide-labeled enriched glycoprotein sample containing 150 μg of protein and a mock enriched control (which was labeled with Ac4 GlcNAc, reacted with Phos-FLAG-His6 , and enriched via anti-FLAG and Ni-NTA affinity chromatography, as described above) were concentrated using a Microcon centrifugal filter device (Millipore) to ∼10 μl, suspended in 8 m urea and 100 mm Tris-HCl (pH 8.5), reduced with 5 mm tris (2-carboxyethyl)phosphine (TCEP), and reacted with 10 mm iodoacetamide. .. The samples were diluted by a factor of four with 100 mm Tris-HCl (pH 8.5) and 1 mm CaCl2 , and then digested with 0.25 μg of trypsin (Promega) overnight at 37 °C.

Lysis:

Article Title: Spatial specificity of auxin responses coordinates wood formation
Article Snippet: .. The transfected protoplasts were diluted with 240 mM CaCl2 (1:3) followed by cell lysis and dual-luciferase assay using the Dual-Luciferase Reporter Assay System (Promega) and following the manufacturer’s instructions. .. Luminescence was measured using a Synergy H4 Hybrid Multiplate Reader (BioTek).

Concentration Assay:

Article Title: Novel Citronellyl-Based Photoprobes Designed to Identify ER Proteins Interacting with Dolichyl Phosphate in Yeast and Mammalian Cells
Article Snippet: Solubilized samples were diluted with 5 mL PBS to 0.2% SDS final concentration. .. After washing with PBS, the beads were resuspended in 200 μl 2M urea/PBS supplemented with CaCl2 (1 mM) for on-bead digestion with trypsin (Promega, 2 μg per 0.2 mL reaction) at 37°C overnight.

Article Title: The WHHERE coactivator complex is required for retinoic acid-dependent regulation of embryonic symmetry
Article Snippet: After 30 min at room temperature, freshly made 0.5 M IAM (Iodoacetamide, Sigma) was added to a final concentration of 10 mM, and the samples were left at room temperature for another 30 min in the dark. .. Urea was then diluted to 2 M with 0.1 M Tris-HCl pH 8.5, CaCl2 was added to 0.5 mM and modified trypsin (Promega), 1:100 (wt/wt), was added for over 12 h at 37 °C.

Article Title: Human Family with Sequence Similarity 60 Member A (FAM60A) Protein: a New Subunit of the Sin3 Deacetylase Complex *
Article Snippet: After 30 min at room temperature, freshly made 0.5 m IAM (Iodoacetamide, Sigma) was added to a final concentration of 10 m m , and the samples were left at room temperature for another 30 min in the dark. .. Urea was then diluted to 2 m with 0.1 m Tris-HCl, pH 8.5, CaCl2 was added to 0.5 m m , and modified trypsin (Promega), 1:100 (wt/wt), was added for over 12 h at 37 °C.

Article Title: Targeted Identification of Glycosylated Proteins in the Gastric Pathogen Helicobacter pylori (Hp) *
Article Snippet: The samples were diluted by a factor of four with 100 mm Tris-HCl (pH 8.5) and 1 mm CaCl2 , and then digested with 0.25 μg of trypsin (Promega) overnight at 37 °C. .. The digests were mixed with formic acid to a final concentration of 5%, desalted using C18 Spec tips (Varian), fully dried by speed vacuum at room temperature, and analyzed by multidimensional protein identification technology (mudPIT) ( ).

Article Title: Coordinated responses to individual tumor antigens by IgG antibody and CD8+ T cells following cancer vaccination
Article Snippet: .. In brief, samples were dried, dissolved in 10 μL of 4X buffer (8 M urea,1 M Tris (pH 8.5), 8 mM CaCl2 , 0.2 M methylamine), reduced, alkylated, diluted to a final 2 M urea concentration and digested by addition of 1.6 μg of sequencing grade trypsin overnight (ProMega) Completion of the digestion was confirmed by 1-D gel analysis. .. Twenty-five μg of each digested sample was then solid phase extracted using Oasis HLB 1cm3 cartridges (Waters Corporation), and peptides dried by vacuum centrifugation.

SDS Page:

Article Title: Raf Kinase Inhibitor Protein (RKIP) Blocks Signal Transducer and Activator of Transcription 3 (STAT3) Activation in Breast and Prostate Cancer
Article Snippet: Identification of Proteins Interacting with RKIP via Liquid Chromatography-Mass Spectrometry (LC-MS/MS) Cells were transfected with HA-RKIP for 48 h. Lysed cells were immunoprecipitated with an antibody to HA and proteins were separated via 10% SDS-PAGE. .. After rehydratation for 15 minutes at 4°C in digestion buffer consisting of 0.05 M ammonium bicarbonate, 5 mM CaCl2 and 12.5 μg/mL trypsin (porcine, sequence grade, Promega, Madison, WI, USA), the proteins were digested.

Plasmid Preparation:

Article Title: Calcium Depletion Dissociates and Activates Heterodimeric Notch Receptors
Article Snippet: NIH 3T3 cells growing in six-well dishes were cotransfected in triplicate with either 1 μg of the HES-AB-luciferase or 1 μg of the HES-ΔAB-luciferase plasmids containing hairy/enhancer of split 1 (HES1) promoter elements ( ) and 20 ng of a Renilla luciferase control plasmid (pRL-TK; Promega) by using Lipofectamine Plus reagent (Gibco-BRL). .. At 24 h after transfection, the cells were briefly treated with HBS containing various concentrations of EDTA or 2.5 mM CaCl2 as described above and then allowed to recover in D10 for up to 8 h. Firefly and Renilla luciferase activities were determined in whole cell extracts using the Dual Luciferase Assay Kit (Promega) and a Turner Designs TD20 dual luminometer.

Irradiation:

Article Title: An allosteric propofol-binding site in kinesin disrupts kinesin-mediated processive movement on microtubules
Article Snippet: Samples were then exposed to UV irradiation, and 20 μg of protein was precipitated using 4 volumes of chilled acetone, and proteins were precipitated overnight at −20 °C. .. Subsequently, 1 μl of 0.1 m CaCl2 and 1 μl of 1% ProteaseMaxTM Surfactant Enhancer (w/v%) was added, and proteins were digested overnight at 37 °C using sequencing grade trypsin (Promega) at ∼1:20 protease:protein (w/w) ratio.

Selection:

Article Title: Coordinated responses to individual tumor antigens by IgG antibody and CD8+ T cells following cancer vaccination
Article Snippet: In brief, samples were dried, dissolved in 10 μL of 4X buffer (8 M urea,1 M Tris (pH 8.5), 8 mM CaCl2 , 0.2 M methylamine), reduced, alkylated, diluted to a final 2 M urea concentration and digested by addition of 1.6 μg of sequencing grade trypsin overnight (ProMega) Completion of the digestion was confirmed by 1-D gel analysis. .. Peptides were analyzed using an Orbitrap Fusion Mass Spectrometer (Thermo Scientific) with a synchronous precursor selection MS3 TMT method [ ].

Sample Prep:

Article Title: Raf Kinase Inhibitor Protein (RKIP) Blocks Signal Transducer and Activator of Transcription 3 (STAT3) Activation in Breast and Prostate Cancer
Article Snippet: Sample preparation, including protein alkylation before proteolytic digestion, was performed as previously described . .. After rehydratation for 15 minutes at 4°C in digestion buffer consisting of 0.05 M ammonium bicarbonate, 5 mM CaCl2 and 12.5 μg/mL trypsin (porcine, sequence grade, Promega, Madison, WI, USA), the proteins were digested.

In Vitro:

Article Title: Both Ser361 phosphorylation and the C‐arrestin domain of thioredoxin interacting protein are important for cell cycle blockade at the G1/S checkpoint
Article Snippet: Paragraph title: In vitro kinase assay ... For the PKC reaction, 20 mm Tris/HCl pH 7.5, 5 mm MgCl2 , 1 mm CaCl2 , 320 ng·μL−1 phosphatidylserine, 32 ng·μL−1 diacylglycerol, and 3 ng·μL−1 PKC (Promega) were added to the kinase reaction mix.

Article Title: Assembly and Function of the RNA Editing Complex in Trypanosoma brucei Requires Band III Protein
Article Snippet: Paragraph title: Extract preparation, adenylylation, and in vitro editing reactions. ... U deletion reactions were in 10 mM KCl-MRB (25 mM Tris-HCl [pH 7.5], 10 mM magnesium acetate, 10 mM KCl, 1mM EDTA, 0.5 mM dithiothreitol [DTT], 5% glycerol) supplemented with 0.15 mM ATP, 3 mM ADP, 5 mM CaCl2 , 4.5 ng of torula RNA per μl, 5 mM DTT, 0.8 U of RNasin per ml (Promega).

Activation Assay:

Article Title: Calcium Depletion Dissociates and Activates Heterodimeric Notch Receptors
Article Snippet: Paragraph title: Transcriptional activation assays. ... At 24 h after transfection, the cells were briefly treated with HBS containing various concentrations of EDTA or 2.5 mM CaCl2 as described above and then allowed to recover in D10 for up to 8 h. Firefly and Renilla luciferase activities were determined in whole cell extracts using the Dual Luciferase Assay Kit (Promega) and a Turner Designs TD20 dual luminometer.

Staining:

Article Title: Raf Kinase Inhibitor Protein (RKIP) Blocks Signal Transducer and Activator of Transcription 3 (STAT3) Activation in Breast and Prostate Cancer
Article Snippet: The gel was stained with coomasie and the gel bands were excised by extracting gel particles with glass Pasteur pipettes. .. After rehydratation for 15 minutes at 4°C in digestion buffer consisting of 0.05 M ammonium bicarbonate, 5 mM CaCl2 and 12.5 μg/mL trypsin (porcine, sequence grade, Promega, Madison, WI, USA), the proteins were digested.

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  • 93
    Promega dnase i buffer
    Profiles of chromatin DNase I-resistance across the 60D cluster region and surrounding sequences. Chromatin resistance to <t>DNase</t> I [as normalized relative yield (NRY) for each amplicon] is plotted on the vertical axis; the length of 60D1-2 genomic region (in kb) is plotted on the horizontal. Positions of the genes in the region are shown at bottom. Circles (black for the regulated chromatin domain, and white for the rest of the region) indicate average NRY for each amplicon, the grey area corresponds to the calculated 95% confidence interval. Upper panel: in larval testes, the entire region shows nearly uniformal low resistance to DNase I typical for the ‘open’ chromatin. In contrast, in larval brains (middle panel) and in embryos (lower panel) regulated chromatin domain that contains the genes CG13589 through Pros28.1B shows significantly higher resistance to DNase I, indicative of ‘closed’ chromatin configuration.
    Dnase I Buffer, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dnase i buffer/product/Promega
    Average 93 stars, based on 6 article reviews
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    dnase i buffer - by Bioz Stars, 2020-02
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    93
    Promega thermolysin digestion buffer
    EBOV-515 and -520 Are Specific to the Base Region of GP and Possess a Capacity to Inhibit GP Cleavage (A and B) Identification of major antigenic sites for three bNAbs using a competition binding assay with intact Jurkat-EBOV GP (A) or <t>thermolysin-cleaved</t> Jurkat-EBOV GP CL (B). Cells were incubated with the first unlabeled mAb and then with the second fluorescently labeled mAb. Binding of mAbs was analyzed by flow cytometry. Numbers indicate the percent binding of the second fluorescently labeled mAb in the presence of the first unlabeled mAb, compared to binding of the second mAb alone. (C) Capacity of bound mAbs to inhibit the exposure of the RBS after EBOV GP to EBOV GP CL . Varying concentrations of mAbs (1, 10, 20, or 40 μg/mL) were incubated with Jurkat-EBOV GP, followed by cleavage and measurement of the exposure of the RBS with fluorescently labeled RBS-specific mAb MR78 by flow cytometric analysis. Dotted line indicates % RBS exposure in the presence of control mAb DENV 2D22. (D) Stability of mAb binding to Jurkat-EBOV GP or Jurkat-EBOV GP CL at neutral or acidic pH. Cells displaying GP or GP CL on the surface were fixed, pre-incubated with fluorescently labeled mAb at neutral pH, and then exposed to neutral or low pH for 60 min. mAb binding was assessed by flow cytometry. Stability of binding was expressed as the percent of the control (maximal binding) when cells were analyzed immediately after staining and without exposure to the neutral or low pH. Mean ± SD of triplicates are shown, and data are representative of 2–3 independent experiments. See also Figure S4 .
    Thermolysin Digestion Buffer, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/thermolysin digestion buffer/product/Promega
    Average 93 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    thermolysin digestion buffer - by Bioz Stars, 2020-02
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    Image Search Results


    Profiles of chromatin DNase I-resistance across the 60D cluster region and surrounding sequences. Chromatin resistance to DNase I [as normalized relative yield (NRY) for each amplicon] is plotted on the vertical axis; the length of 60D1-2 genomic region (in kb) is plotted on the horizontal. Positions of the genes in the region are shown at bottom. Circles (black for the regulated chromatin domain, and white for the rest of the region) indicate average NRY for each amplicon, the grey area corresponds to the calculated 95% confidence interval. Upper panel: in larval testes, the entire region shows nearly uniformal low resistance to DNase I typical for the ‘open’ chromatin. In contrast, in larval brains (middle panel) and in embryos (lower panel) regulated chromatin domain that contains the genes CG13589 through Pros28.1B shows significantly higher resistance to DNase I, indicative of ‘closed’ chromatin configuration.

    Journal: Nucleic Acids Research

    Article Title: Regulated chromatin domain comprising cluster of co-expressed genes in Drosophila melanogaster

    doi: 10.1093/nar/gki281

    Figure Lengend Snippet: Profiles of chromatin DNase I-resistance across the 60D cluster region and surrounding sequences. Chromatin resistance to DNase I [as normalized relative yield (NRY) for each amplicon] is plotted on the vertical axis; the length of 60D1-2 genomic region (in kb) is plotted on the horizontal. Positions of the genes in the region are shown at bottom. Circles (black for the regulated chromatin domain, and white for the rest of the region) indicate average NRY for each amplicon, the grey area corresponds to the calculated 95% confidence interval. Upper panel: in larval testes, the entire region shows nearly uniformal low resistance to DNase I typical for the ‘open’ chromatin. In contrast, in larval brains (middle panel) and in embryos (lower panel) regulated chromatin domain that contains the genes CG13589 through Pros28.1B shows significantly higher resistance to DNase I, indicative of ‘closed’ chromatin configuration.

    Article Snippet: Pellets were resuspended in the DNase I-buffer (40 mM Tris–HCl, 0.4 mM EDTA, 10 mM MgCl2 , 10 mM CaCl2 and 0.1 mg/ml bovine serum albumin) and treated with 0.5 U/ml DNase I (Promega).

    Techniques: Amplification

    EBOV-515 and -520 Are Specific to the Base Region of GP and Possess a Capacity to Inhibit GP Cleavage (A and B) Identification of major antigenic sites for three bNAbs using a competition binding assay with intact Jurkat-EBOV GP (A) or thermolysin-cleaved Jurkat-EBOV GP CL (B). Cells were incubated with the first unlabeled mAb and then with the second fluorescently labeled mAb. Binding of mAbs was analyzed by flow cytometry. Numbers indicate the percent binding of the second fluorescently labeled mAb in the presence of the first unlabeled mAb, compared to binding of the second mAb alone. (C) Capacity of bound mAbs to inhibit the exposure of the RBS after EBOV GP to EBOV GP CL . Varying concentrations of mAbs (1, 10, 20, or 40 μg/mL) were incubated with Jurkat-EBOV GP, followed by cleavage and measurement of the exposure of the RBS with fluorescently labeled RBS-specific mAb MR78 by flow cytometric analysis. Dotted line indicates % RBS exposure in the presence of control mAb DENV 2D22. (D) Stability of mAb binding to Jurkat-EBOV GP or Jurkat-EBOV GP CL at neutral or acidic pH. Cells displaying GP or GP CL on the surface were fixed, pre-incubated with fluorescently labeled mAb at neutral pH, and then exposed to neutral or low pH for 60 min. mAb binding was assessed by flow cytometry. Stability of binding was expressed as the percent of the control (maximal binding) when cells were analyzed immediately after staining and without exposure to the neutral or low pH. Mean ± SD of triplicates are shown, and data are representative of 2–3 independent experiments. See also Figure S4 .

    Journal: Immunity

    Article Title: Multifunctional Pan-ebolavirus Antibody Recognizes a Site of Broad Vulnerability on the Ebolavirus Glycoprotein

    doi: 10.1016/j.immuni.2018.06.018

    Figure Lengend Snippet: EBOV-515 and -520 Are Specific to the Base Region of GP and Possess a Capacity to Inhibit GP Cleavage (A and B) Identification of major antigenic sites for three bNAbs using a competition binding assay with intact Jurkat-EBOV GP (A) or thermolysin-cleaved Jurkat-EBOV GP CL (B). Cells were incubated with the first unlabeled mAb and then with the second fluorescently labeled mAb. Binding of mAbs was analyzed by flow cytometry. Numbers indicate the percent binding of the second fluorescently labeled mAb in the presence of the first unlabeled mAb, compared to binding of the second mAb alone. (C) Capacity of bound mAbs to inhibit the exposure of the RBS after EBOV GP to EBOV GP CL . Varying concentrations of mAbs (1, 10, 20, or 40 μg/mL) were incubated with Jurkat-EBOV GP, followed by cleavage and measurement of the exposure of the RBS with fluorescently labeled RBS-specific mAb MR78 by flow cytometric analysis. Dotted line indicates % RBS exposure in the presence of control mAb DENV 2D22. (D) Stability of mAb binding to Jurkat-EBOV GP or Jurkat-EBOV GP CL at neutral or acidic pH. Cells displaying GP or GP CL on the surface were fixed, pre-incubated with fluorescently labeled mAb at neutral pH, and then exposed to neutral or low pH for 60 min. mAb binding was assessed by flow cytometry. Stability of binding was expressed as the percent of the control (maximal binding) when cells were analyzed immediately after staining and without exposure to the neutral or low pH. Mean ± SD of triplicates are shown, and data are representative of 2–3 independent experiments. See also Figure S4 .

    Article Snippet: Pelleted virus was resuspended in thermolysin digestion buffer (50 mM Tris, 0.5 mM CaCl2 pH 8.0) and divided into 2 aliquots: one aliquot was treated with 0.5 mg/mL of thermolysin (Promega), another one – with equal volume of thermolysin digestion buffer (mock-treated virus) for 40 min at 37°C.

    Techniques: Binding Assay, Incubation, Labeling, Flow Cytometry, Cytometry, Staining

    EBOV-515 and -520 Are Specific to the Base Region of GP and Possess a Capacity to Inhibit GP Cleavage (A and B) Identification of major antigenic sites for three bNAbs using a competition binding assay with intact Jurkat-EBOV GP (A) or thermolysin-cleaved Jurkat-EBOV GP CL (B). Cells were incubated with the first unlabeled mAb and then with the second fluorescently labeled mAb. Binding of mAbs was analyzed by flow cytometry. Numbers indicate the percent binding of the second fluorescently labeled mAb in the presence of the first unlabeled mAb, compared to binding of the second mAb alone. (C) Capacity of bound mAbs to inhibit the exposure of the RBS after EBOV GP to EBOV GP CL . Varying concentrations of mAbs (1, 10, 20, or 40 μg/mL) were incubated with Jurkat-EBOV GP, followed by cleavage and measurement of the exposure of the RBS with fluorescently labeled RBS-specific mAb MR78 by flow cytometric analysis. Dotted line indicates % RBS exposure in the presence of control mAb DENV 2D22. (D) Stability of mAb binding to Jurkat-EBOV GP or Jurkat-EBOV GP CL at neutral or acidic pH. Cells displaying GP or GP CL on the surface were fixed, pre-incubated with fluorescently labeled mAb at neutral pH, and then exposed to neutral or low pH for 60 min. mAb binding was assessed by flow cytometry. Stability of binding was expressed as the percent of the control (maximal binding) when cells were analyzed immediately after staining and without exposure to the neutral or low pH. .

    Journal: Immunity

    Article Title: Multifunctional Pan-ebolavirus Antibody Recognizes a Site of Broad Vulnerability on the Ebolavirus Glycoprotein

    doi: 10.1016/j.immuni.2018.06.018

    Figure Lengend Snippet: EBOV-515 and -520 Are Specific to the Base Region of GP and Possess a Capacity to Inhibit GP Cleavage (A and B) Identification of major antigenic sites for three bNAbs using a competition binding assay with intact Jurkat-EBOV GP (A) or thermolysin-cleaved Jurkat-EBOV GP CL (B). Cells were incubated with the first unlabeled mAb and then with the second fluorescently labeled mAb. Binding of mAbs was analyzed by flow cytometry. Numbers indicate the percent binding of the second fluorescently labeled mAb in the presence of the first unlabeled mAb, compared to binding of the second mAb alone. (C) Capacity of bound mAbs to inhibit the exposure of the RBS after EBOV GP to EBOV GP CL . Varying concentrations of mAbs (1, 10, 20, or 40 μg/mL) were incubated with Jurkat-EBOV GP, followed by cleavage and measurement of the exposure of the RBS with fluorescently labeled RBS-specific mAb MR78 by flow cytometric analysis. Dotted line indicates % RBS exposure in the presence of control mAb DENV 2D22. (D) Stability of mAb binding to Jurkat-EBOV GP or Jurkat-EBOV GP CL at neutral or acidic pH. Cells displaying GP or GP CL on the surface were fixed, pre-incubated with fluorescently labeled mAb at neutral pH, and then exposed to neutral or low pH for 60 min. mAb binding was assessed by flow cytometry. Stability of binding was expressed as the percent of the control (maximal binding) when cells were analyzed immediately after staining and without exposure to the neutral or low pH. .

    Article Snippet: Pelleted virus was resuspended in thermolysin digestion buffer (50 mM Tris, 0.5 mM CaCl2 pH 8.0) and divided into 2 aliquots: one aliquot was treated with 0.5 mg/mL of thermolysin (Promega), another one – with equal volume of thermolysin digestion buffer (mock-treated virus) for 40 min at 37°C.

    Techniques: Binding Assay, Incubation, Labeling, Flow Cytometry, Cytometry, Staining

    Stalk- and glycan cap-specific mAbs suppress viral infection by different mechanisms. A. MPER-specific, but not glycan cap-specific mAbs neutralize thermolysin-treated virus. Bars represent fold reduction of viral titers upon incubation with mAbs, mean values of triplicate samples ± SE. P values were calculated by unpaired Student’s t-test. Western blot shows elimination of the band corresponding to intact GP1 subunit after treatment of VSV/BDBV-GP particles with thermolysin. B. MAbs do not prevent cathepsin cleavage of GP. VLPs were incubated with indicated mAbs, treated with cathepsins B/L or mock-treated where indicated, and separated on acrylamide gel. The 20 kDa bands corresponding to cleavage product are shown. Numbers below the western blots represent GP values normalized to VP40 values. C. Inhibition of cell-to-cell virus transmission by mAbs. Top, schematic representation of the assay procedure. Bottom, assay results. Bars indicate percent of CellTrace Far Red + /eGFP + cells out of total CellTrace Far Red + population normalized to no mAb control, mean values of triplicate samples ± SE. P values were calculated by unpaired Student’s t-test, compared to no mAb control. The mAb concentrations are indicated in panel D. D. Glycan cap-specific mAbs are efficient inhibitors of virus egress. Bars indicate viral RNA load, determined by digital droplet RT-PCR, in the supernatants of cells infected with EBOV/BDBV-GP normalized to no mAb control, mean values of triplicate samples ± SE. P values were calculated by unpaired Student’s t-test, compared to no mAb control. E. MPER-specific mAbs are more effective than glycan cap-specific mAbs when added after infection. MAbs were added at different time points after inoculation of cells with EBOV/BDBV-GP. At 48 hours after inoculation, cells were fixed and analyzed by flow cytometry. Bars indicate percentages of eGFP + cells normalized to no mAb control, mean values of triplicate samples ± SE. P values were calculated by unpaired Student’s t- test, compared to no mAb control.

    Journal: PLoS Pathogens

    Article Title: Asymmetric antiviral effects of ebolavirus antibodies targeting glycoprotein stem and glycan cap

    doi: 10.1371/journal.ppat.1007204

    Figure Lengend Snippet: Stalk- and glycan cap-specific mAbs suppress viral infection by different mechanisms. A. MPER-specific, but not glycan cap-specific mAbs neutralize thermolysin-treated virus. Bars represent fold reduction of viral titers upon incubation with mAbs, mean values of triplicate samples ± SE. P values were calculated by unpaired Student’s t-test. Western blot shows elimination of the band corresponding to intact GP1 subunit after treatment of VSV/BDBV-GP particles with thermolysin. B. MAbs do not prevent cathepsin cleavage of GP. VLPs were incubated with indicated mAbs, treated with cathepsins B/L or mock-treated where indicated, and separated on acrylamide gel. The 20 kDa bands corresponding to cleavage product are shown. Numbers below the western blots represent GP values normalized to VP40 values. C. Inhibition of cell-to-cell virus transmission by mAbs. Top, schematic representation of the assay procedure. Bottom, assay results. Bars indicate percent of CellTrace Far Red + /eGFP + cells out of total CellTrace Far Red + population normalized to no mAb control, mean values of triplicate samples ± SE. P values were calculated by unpaired Student’s t-test, compared to no mAb control. The mAb concentrations are indicated in panel D. D. Glycan cap-specific mAbs are efficient inhibitors of virus egress. Bars indicate viral RNA load, determined by digital droplet RT-PCR, in the supernatants of cells infected with EBOV/BDBV-GP normalized to no mAb control, mean values of triplicate samples ± SE. P values were calculated by unpaired Student’s t-test, compared to no mAb control. E. MPER-specific mAbs are more effective than glycan cap-specific mAbs when added after infection. MAbs were added at different time points after inoculation of cells with EBOV/BDBV-GP. At 48 hours after inoculation, cells were fixed and analyzed by flow cytometry. Bars indicate percentages of eGFP + cells normalized to no mAb control, mean values of triplicate samples ± SE. P values were calculated by unpaired Student’s t- test, compared to no mAb control.

    Article Snippet: VSV/BDBV-GP purified as described above was resuspended in thermolysin digestion buffer (50 mM Tris, pH 8.0, 0.5 mM CaCl2 ) and divided into two aliquots; one aliquot was treated with 0.5 mg/ml of thermolysin (Promega) and another one with an equal volume of thermolysin digestion buffer (mock-treated virus) for 40 min at 37ºC.

    Techniques: Infection, Incubation, Western Blot, Acrylamide Gel Assay, Inhibition, Transmission Assay, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Cytometry