Structured Review

Promega cacl2
Cacl2, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 113 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/cacl2/product/Promega
Average 94 stars, based on 113 article reviews
Price from $9.99 to $1999.99
cacl2 - by Bioz Stars, 2020-09
94/100 stars

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Sequencing:

Article Title: Identification of E-cadherin signature motifs functioning as cleavage sites for Helicobacter pylori HtrA
Article Snippet: .. Finally, 2 μl of sequencing-grade modified porcine trypsin was added (stock solution of 50 ng/μl in 50 mM HEPES, 1 mM CaCl2 , pH 7.3, Promega). ..

Article Title: Structural analyses of the chromatin remodeling enzymes INO80-C and SWR-C
Article Snippet: .. Protein samples were then digested using sequencing grade Endoproteinase Lys-C (Roche, Cat#11047817001) (0.5 µg) for 6 h at 37°C, diluted with 100 mM Tris, pH 8.5, to reduce the concentration of urea to 2 M, supplemented with 2 mM CaCl2 and incubated overnight with sequencing grade Trypsin protease (Promega Cat#V5111) (0.5 µg) at 37°C. .. Reactions were quenched by adding formic acid (J.T.Baker, Cat#0129-01) to a final concentration of 5% (v/v).

Article Title: CLIMP-63 is a gentamicin-binding protein that is involved in drug-induced cytotoxicity
Article Snippet: .. Mass spectrometry Gentamicin-binding protein bands from KPT11 cells were excised from Coomassie-stained gels, washed twice for 30 min in 50 mM ammonium bicarbonate/50% (v/v) acetonitrile, reduced in 10 mM DTT/100 mM ammonium bicarbonate, alkylated in 55 mM iodoacetamide/100 mM ammonium bicarbonate, and dehydrated gels reswelled in digestion buffer containing 50 mM ammonium bicarbonate/5 mM CaCl2 /12.5 ng/μ l sequencing grade modified trypsin (Promega, Madison, WI, USA). .. Following an overnight incubation at 37°C, extracted peptides were identified by collection of MS/MS data using either a LTQ linear ion trap mass spectrometer (ThermoFisher, San Jose, CA, USA) or a Q-Star XL hybrid quadrupole-TOF mass spectrometer (Applied Biosystems, Foster City, CA, USA).

Article Title: Proteomic analysis reveals metabolic and regulatory systems involved in the syntrophic and axenic lifestyle of Syntrophomonas wolfei
Article Snippet: .. Lysates were cooled to ambient temperature, and diluted with 50 mM Tris with 10 mM CaCl2 to decrease the guanidine concentration to ~1 M. Ten micrograms of trypsin (sequencing grade, Promega, Madison WI) was added to each lysate, followed by a 5-h incubation at 37°C. .. Remaining disulfide bonds were reduced by adding additional DTT to a final concentration of 10 mM and incubation for 1 h at 37°C.

Article Title: Encoding human serine phosphopeptides in bacteria for proteome-wide identification of phosphorylation-dependent interactions
Article Snippet: .. 7 μL of 200 mM DTT was then added to quench the reaction, and then 1.1 mL water, 6.7 μL CaCl2, 133 μL 1M Tris pH 8.5 and 60 μL 0.5 mg/mL sequence-grade trypsin (Promega) were added to each sample. .. Surfactant was then cleaved using 125 μL 20% trifluoroacetic acid and incubating samples at room temperature for 15 min. Peptides were desalted using two C18 silica MicroSpin Columns (The Nest Group) in sequence, and eluted in 300 μL 80% acetonitrile 0.1% trifluoroacetic acid and dried by centrifugal vacuum.

Article Title: Comparative Community Proteomics Demonstrates the Unexpected Importance of Actinobacterial Glycoside Hydrolase Family 12 Protein for Crystalline Cellulose Hydrolysis
Article Snippet: .. Samples were then diluted 8-fold for preparation for digestion with 100 mM NH4 HCO3 –1 mM CaCl2 , and sequencing-grade modified porcine trypsin (Promega, Madison, WI) was added to all protein samples at a 1:50 (wt/wt) trypsin/protein ratio for 3 h at 37°C. .. Digested samples were desalted using a 4-probe positive-pressure Gilson GX-274 ASPEC system (Gilson, Inc., Middleton, WI) with Discovery C18 100-mg/ml solid-phase extraction tubes (Supelco, St. Louis, MO) as previously described ( ).

Concentration Assay:

Article Title: Structural analyses of the chromatin remodeling enzymes INO80-C and SWR-C
Article Snippet: .. Protein samples were then digested using sequencing grade Endoproteinase Lys-C (Roche, Cat#11047817001) (0.5 µg) for 6 h at 37°C, diluted with 100 mM Tris, pH 8.5, to reduce the concentration of urea to 2 M, supplemented with 2 mM CaCl2 and incubated overnight with sequencing grade Trypsin protease (Promega Cat#V5111) (0.5 µg) at 37°C. .. Reactions were quenched by adding formic acid (J.T.Baker, Cat#0129-01) to a final concentration of 5% (v/v).

Article Title: Proteomic analysis reveals metabolic and regulatory systems involved in the syntrophic and axenic lifestyle of Syntrophomonas wolfei
Article Snippet: .. Lysates were cooled to ambient temperature, and diluted with 50 mM Tris with 10 mM CaCl2 to decrease the guanidine concentration to ~1 M. Ten micrograms of trypsin (sequencing grade, Promega, Madison WI) was added to each lysate, followed by a 5-h incubation at 37°C. .. Remaining disulfide bonds were reduced by adding additional DTT to a final concentration of 10 mM and incubation for 1 h at 37°C.

Incubation:

Article Title: Structural analyses of the chromatin remodeling enzymes INO80-C and SWR-C
Article Snippet: .. Protein samples were then digested using sequencing grade Endoproteinase Lys-C (Roche, Cat#11047817001) (0.5 µg) for 6 h at 37°C, diluted with 100 mM Tris, pH 8.5, to reduce the concentration of urea to 2 M, supplemented with 2 mM CaCl2 and incubated overnight with sequencing grade Trypsin protease (Promega Cat#V5111) (0.5 µg) at 37°C. .. Reactions were quenched by adding formic acid (J.T.Baker, Cat#0129-01) to a final concentration of 5% (v/v).

Article Title: Proteomic analysis reveals metabolic and regulatory systems involved in the syntrophic and axenic lifestyle of Syntrophomonas wolfei
Article Snippet: .. Lysates were cooled to ambient temperature, and diluted with 50 mM Tris with 10 mM CaCl2 to decrease the guanidine concentration to ~1 M. Ten micrograms of trypsin (sequencing grade, Promega, Madison WI) was added to each lysate, followed by a 5-h incubation at 37°C. .. Remaining disulfide bonds were reduced by adding additional DTT to a final concentration of 10 mM and incubation for 1 h at 37°C.

Mass Spectrometry:

Article Title: CLIMP-63 is a gentamicin-binding protein that is involved in drug-induced cytotoxicity
Article Snippet: .. Mass spectrometry Gentamicin-binding protein bands from KPT11 cells were excised from Coomassie-stained gels, washed twice for 30 min in 50 mM ammonium bicarbonate/50% (v/v) acetonitrile, reduced in 10 mM DTT/100 mM ammonium bicarbonate, alkylated in 55 mM iodoacetamide/100 mM ammonium bicarbonate, and dehydrated gels reswelled in digestion buffer containing 50 mM ammonium bicarbonate/5 mM CaCl2 /12.5 ng/μ l sequencing grade modified trypsin (Promega, Madison, WI, USA). .. Following an overnight incubation at 37°C, extracted peptides were identified by collection of MS/MS data using either a LTQ linear ion trap mass spectrometer (ThermoFisher, San Jose, CA, USA) or a Q-Star XL hybrid quadrupole-TOF mass spectrometer (Applied Biosystems, Foster City, CA, USA).

Modification:

Article Title: Identification of E-cadherin signature motifs functioning as cleavage sites for Helicobacter pylori HtrA
Article Snippet: .. Finally, 2 μl of sequencing-grade modified porcine trypsin was added (stock solution of 50 ng/μl in 50 mM HEPES, 1 mM CaCl2 , pH 7.3, Promega). ..

Article Title: CLIMP-63 is a gentamicin-binding protein that is involved in drug-induced cytotoxicity
Article Snippet: .. Mass spectrometry Gentamicin-binding protein bands from KPT11 cells were excised from Coomassie-stained gels, washed twice for 30 min in 50 mM ammonium bicarbonate/50% (v/v) acetonitrile, reduced in 10 mM DTT/100 mM ammonium bicarbonate, alkylated in 55 mM iodoacetamide/100 mM ammonium bicarbonate, and dehydrated gels reswelled in digestion buffer containing 50 mM ammonium bicarbonate/5 mM CaCl2 /12.5 ng/μ l sequencing grade modified trypsin (Promega, Madison, WI, USA). .. Following an overnight incubation at 37°C, extracted peptides were identified by collection of MS/MS data using either a LTQ linear ion trap mass spectrometer (ThermoFisher, San Jose, CA, USA) or a Q-Star XL hybrid quadrupole-TOF mass spectrometer (Applied Biosystems, Foster City, CA, USA).

Article Title: Comparative Community Proteomics Demonstrates the Unexpected Importance of Actinobacterial Glycoside Hydrolase Family 12 Protein for Crystalline Cellulose Hydrolysis
Article Snippet: .. Samples were then diluted 8-fold for preparation for digestion with 100 mM NH4 HCO3 –1 mM CaCl2 , and sequencing-grade modified porcine trypsin (Promega, Madison, WI) was added to all protein samples at a 1:50 (wt/wt) trypsin/protein ratio for 3 h at 37°C. .. Digested samples were desalted using a 4-probe positive-pressure Gilson GX-274 ASPEC system (Gilson, Inc., Middleton, WI) with Discovery C18 100-mg/ml solid-phase extraction tubes (Supelco, St. Louis, MO) as previously described ( ).

Chloramphenicol Acetyltransferase Assay:

Article Title: Structural analyses of the chromatin remodeling enzymes INO80-C and SWR-C
Article Snippet: .. Protein samples were then digested using sequencing grade Endoproteinase Lys-C (Roche, Cat11047817001) (0.5 µg) for 6 h at 37°C, diluted with 100 mM Tris, pH 8.5, to reduce the concentration of urea to 2 M, supplemented with 2 mM CaCl2 and incubated overnight with sequencing grade Trypsin protease (Promega Cat#V5111) (0.5 µg) at 37°C. .. Reactions were quenched by adding formic acid (J.T.Baker, Cat#0129-01) to a final concentration of 5% (v/v).

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  • 92
    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: 92/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dnase i buffer/product/Promega
    Average 92 stars, based on 11 article reviews
    Price from $9.99 to $1999.99
    dnase i buffer - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    99
    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: 99/100, 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 99 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    thermolysin digestion buffer - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    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

    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: Neutralization of thermolysin-treated VSV/BDBV-GP by mAbs 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

    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