con a sepharose  (GE Healthcare)

 
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    Name:
    Con A Sepharose 4B
    Description:

    Catalog Number:
    17044001
    Price:
    None
    Category:
    Con A Sepharose 4B
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    Structured Review

    GE Healthcare con a sepharose
    Lipid remodeling of lipid moieties in GPI anchors to ceramides is defective in cwh43 Δ cells. (A) Analysis of PI moieties obtained from GPI-anchored proteins in wild-type (WT), per1 Δ, gup1 Δ, per1 Δ gup1 Δ, gpi7 Δ, and cwh43 Δ cells. Cells were labeled with [ 3 H]inositol at 30°C. Cell lysate was delipidated, and glycoproteins were concentrated using Con <t>A-Sepharose.</t> PI moieties of GPI anchors were released by deamination by using nitrous acid, separated by TLC by using solvent system 1, detected by autoradiography, and analyzed using a Molecular Imager FX. (B) Analysis of incorporation of [ 3 H]DHS derivatives in proteins. Cells were incubated for 20 min in the presence of myriocin and then labeled with [ 3 H]DHS at 30°C. Glycoproteins were concentrated from extracted lysate using Con A-Sepharose and separated by SDS-PAGE. Radiolabeled proteins were visualized using a Molecular Imager FX. Equal loading of total proteins was confirmed by Coomassie brilliant blue staining of the SDS-PAGE gels.

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    Average 93 stars, based on 1 article reviews
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    con a sepharose - by Bioz Stars, 2021-03
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    Images

    1) Product Images from "Saccharomyces cerevisiae CWH43"

    Article Title: Saccharomyces cerevisiae CWH43

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E07-05-0482

    Lipid remodeling of lipid moieties in GPI anchors to ceramides is defective in cwh43 Δ cells. (A) Analysis of PI moieties obtained from GPI-anchored proteins in wild-type (WT), per1 Δ, gup1 Δ, per1 Δ gup1 Δ, gpi7 Δ, and cwh43 Δ cells. Cells were labeled with [ 3 H]inositol at 30°C. Cell lysate was delipidated, and glycoproteins were concentrated using Con A-Sepharose. PI moieties of GPI anchors were released by deamination by using nitrous acid, separated by TLC by using solvent system 1, detected by autoradiography, and analyzed using a Molecular Imager FX. (B) Analysis of incorporation of [ 3 H]DHS derivatives in proteins. Cells were incubated for 20 min in the presence of myriocin and then labeled with [ 3 H]DHS at 30°C. Glycoproteins were concentrated from extracted lysate using Con A-Sepharose and separated by SDS-PAGE. Radiolabeled proteins were visualized using a Molecular Imager FX. Equal loading of total proteins was confirmed by Coomassie brilliant blue staining of the SDS-PAGE gels.
    Figure Legend Snippet: Lipid remodeling of lipid moieties in GPI anchors to ceramides is defective in cwh43 Δ cells. (A) Analysis of PI moieties obtained from GPI-anchored proteins in wild-type (WT), per1 Δ, gup1 Δ, per1 Δ gup1 Δ, gpi7 Δ, and cwh43 Δ cells. Cells were labeled with [ 3 H]inositol at 30°C. Cell lysate was delipidated, and glycoproteins were concentrated using Con A-Sepharose. PI moieties of GPI anchors were released by deamination by using nitrous acid, separated by TLC by using solvent system 1, detected by autoradiography, and analyzed using a Molecular Imager FX. (B) Analysis of incorporation of [ 3 H]DHS derivatives in proteins. Cells were incubated for 20 min in the presence of myriocin and then labeled with [ 3 H]DHS at 30°C. Glycoproteins were concentrated from extracted lysate using Con A-Sepharose and separated by SDS-PAGE. Radiolabeled proteins were visualized using a Molecular Imager FX. Equal loading of total proteins was confirmed by Coomassie brilliant blue staining of the SDS-PAGE gels.

    Techniques Used: Labeling, Thin Layer Chromatography, Autoradiography, Incubation, SDS Page, Staining

    2) Product Images from "Elution of tightly bound solutes from concanavalin A Sepharose Factors affecting the desorption of cottonmouth venom glycoproteins"

    Article Title: Elution of tightly bound solutes from concanavalin A Sepharose Factors affecting the desorption of cottonmouth venom glycoproteins

    Journal:

    doi: 10.1016/j.chroma.2007.03.126

    Effects of 4 M urea on Con A Sepharose. A virgin Con A Sepharose column (column filters replaced) was subjected to a standard chromatographic experiment with 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside/4 M urea (pH 4.0) as the eluent.
    Figure Legend Snippet: Effects of 4 M urea on Con A Sepharose. A virgin Con A Sepharose column (column filters replaced) was subjected to a standard chromatographic experiment with 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside/4 M urea (pH 4.0) as the eluent.

    Techniques Used:

    Influence of pH on venom glycoprotein and PDE desorbed from Con A Sepharose. Eluents consisted of 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside titrated with NaOH to pH 3.0, 4.0, 5.0, or 6.0. Bradford data and PDE activities have been
    Figure Legend Snippet: Influence of pH on venom glycoprotein and PDE desorbed from Con A Sepharose. Eluents consisted of 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside titrated with NaOH to pH 3.0, 4.0, 5.0, or 6.0. Bradford data and PDE activities have been

    Techniques Used:

    Influence of auxiliary and alternative eluents on glycoprotein and PDE desorption from Con A Sepharose. Auxiliary eluents were added to the standard 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside (pH 4.0) buffer. These included 20% ethylene
    Figure Legend Snippet: Influence of auxiliary and alternative eluents on glycoprotein and PDE desorption from Con A Sepharose. Auxiliary eluents were added to the standard 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside (pH 4.0) buffer. These included 20% ethylene

    Techniques Used:

    Influence of eluent NaCl concentration on glycoprotein and PDE desorption. Increasing eluent NaCl concentrations reduces glycoprotein desorption from Con A Sepharose even after the Bradford and PDE assays are corrected for direct effects on the assay
    Figure Legend Snippet: Influence of eluent NaCl concentration on glycoprotein and PDE desorption. Increasing eluent NaCl concentrations reduces glycoprotein desorption from Con A Sepharose even after the Bradford and PDE assays are corrected for direct effects on the assay

    Techniques Used: Concentration Assay

    Glycoprotein elution profiles with 0-4 pauses introduced during desorption. The 1 mL Con A Sepharose column was equilibrated in 50 mM acetic acid/NaOH/500 mM NaCl/1 mM MnCl 2 /1 mM CaCl 2 /1 mM MgCl 2 (pH 6.0) and desorbed with 100 mM acetic acid/NaOH/1 M
    Figure Legend Snippet: Glycoprotein elution profiles with 0-4 pauses introduced during desorption. The 1 mL Con A Sepharose column was equilibrated in 50 mM acetic acid/NaOH/500 mM NaCl/1 mM MnCl 2 /1 mM CaCl 2 /1 mM MgCl 2 (pH 6.0) and desorbed with 100 mM acetic acid/NaOH/1 M

    Techniques Used:

    3) Product Images from "Elution of tightly bound solutes from concanavalin A Sepharose Factors affecting the desorption of cottonmouth venom glycoproteins"

    Article Title: Elution of tightly bound solutes from concanavalin A Sepharose Factors affecting the desorption of cottonmouth venom glycoproteins

    Journal:

    doi: 10.1016/j.chroma.2007.03.126

    Effects of 4 M urea on Con A Sepharose. A virgin Con A Sepharose column (column filters replaced) was subjected to a standard chromatographic experiment with 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside/4 M urea (pH 4.0) as the eluent.
    Figure Legend Snippet: Effects of 4 M urea on Con A Sepharose. A virgin Con A Sepharose column (column filters replaced) was subjected to a standard chromatographic experiment with 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside/4 M urea (pH 4.0) as the eluent.

    Techniques Used:

    Influence of pH on venom glycoprotein and PDE desorbed from Con A Sepharose. Eluents consisted of 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside titrated with NaOH to pH 3.0, 4.0, 5.0, or 6.0. Bradford data and PDE activities have been
    Figure Legend Snippet: Influence of pH on venom glycoprotein and PDE desorbed from Con A Sepharose. Eluents consisted of 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside titrated with NaOH to pH 3.0, 4.0, 5.0, or 6.0. Bradford data and PDE activities have been

    Techniques Used:

    Influence of auxiliary and alternative eluents on glycoprotein and PDE desorption from Con A Sepharose. Auxiliary eluents were added to the standard 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside (pH 4.0) buffer. These included 20% ethylene
    Figure Legend Snippet: Influence of auxiliary and alternative eluents on glycoprotein and PDE desorption from Con A Sepharose. Auxiliary eluents were added to the standard 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside (pH 4.0) buffer. These included 20% ethylene

    Techniques Used:

    Influence of eluent NaCl concentration on glycoprotein and PDE desorption. Increasing eluent NaCl concentrations reduces glycoprotein desorption from Con A Sepharose even after the Bradford and PDE assays are corrected for direct effects on the assay
    Figure Legend Snippet: Influence of eluent NaCl concentration on glycoprotein and PDE desorption. Increasing eluent NaCl concentrations reduces glycoprotein desorption from Con A Sepharose even after the Bradford and PDE assays are corrected for direct effects on the assay

    Techniques Used: Concentration Assay

    Glycoprotein elution profiles with 0-4 pauses introduced during desorption. The 1 mL Con A Sepharose column was equilibrated in 50 mM acetic acid/NaOH/500 mM NaCl/1 mM MnCl 2 /1 mM CaCl 2 /1 mM MgCl 2 (pH 6.0) and desorbed with 100 mM acetic acid/NaOH/1 M
    Figure Legend Snippet: Glycoprotein elution profiles with 0-4 pauses introduced during desorption. The 1 mL Con A Sepharose column was equilibrated in 50 mM acetic acid/NaOH/500 mM NaCl/1 mM MnCl 2 /1 mM CaCl 2 /1 mM MgCl 2 (pH 6.0) and desorbed with 100 mM acetic acid/NaOH/1 M

    Techniques Used:

    4) Product Images from "The STT3a Subunit Isoform of the Arabidopsis Oligosaccharyltransferase Controls Adaptive Responses to Salt/Osmotic Stress"

    Article Title: The STT3a Subunit Isoform of the Arabidopsis Oligosaccharyltransferase Controls Adaptive Responses to Salt/Osmotic Stress

    Journal: The Plant Cell

    doi: 10.1105/tpc.013862

    stt3a-1 Plants Are Defective in Protein Glycosylation. (A) Glycosylated proteins in crude extracts of wild-type (C24) and stt3a-1 plants were resolved by SDS-PAGE and detected by Con A lectin blot analysis in total proteins (left gel) and Con A binding proteins (CBPs; right gel). A major 65-kD protein (CBP65) in wild-type seedlings is identified by the arrowhead. The Con A blot was exposed longer to visualize a faint 60-kD band detected in the stt3a-1 plant extract. (B) Endoglycosidase H treatment resolves differences in protein glycosylation in wild-type (C24) and stt3a-1 plants. Proteins from crude extracts were absorbed onto Con A–Sepharose (Pharmacia Amersham), and the bound protein was recovered by elution with α-methylmannopyranoside. Fifteen micrograms of CBPs with or without deglycosylation treatment by endoglycosidase H (endoH) was analyzed by SDS-PAGE. Open arrowheads identify bands that are common in wild-type and stt3a-1 CBP fractions after endoglycosidase H treatment. (C) Alignment of the CBP65 N-terminal sequence with the Arabidopsis TGG1 (At5g26000) peptide sequence. Potential N -glycosylation motifs are underlined.
    Figure Legend Snippet: stt3a-1 Plants Are Defective in Protein Glycosylation. (A) Glycosylated proteins in crude extracts of wild-type (C24) and stt3a-1 plants were resolved by SDS-PAGE and detected by Con A lectin blot analysis in total proteins (left gel) and Con A binding proteins (CBPs; right gel). A major 65-kD protein (CBP65) in wild-type seedlings is identified by the arrowhead. The Con A blot was exposed longer to visualize a faint 60-kD band detected in the stt3a-1 plant extract. (B) Endoglycosidase H treatment resolves differences in protein glycosylation in wild-type (C24) and stt3a-1 plants. Proteins from crude extracts were absorbed onto Con A–Sepharose (Pharmacia Amersham), and the bound protein was recovered by elution with α-methylmannopyranoside. Fifteen micrograms of CBPs with or without deglycosylation treatment by endoglycosidase H (endoH) was analyzed by SDS-PAGE. Open arrowheads identify bands that are common in wild-type and stt3a-1 CBP fractions after endoglycosidase H treatment. (C) Alignment of the CBP65 N-terminal sequence with the Arabidopsis TGG1 (At5g26000) peptide sequence. Potential N -glycosylation motifs are underlined.

    Techniques Used: SDS Page, Binding Assay, Sequencing

    5) Product Images from "Elution of tightly bound solutes from concanavalin A Sepharose Factors affecting the desorption of cottonmouth venom glycoproteins"

    Article Title: Elution of tightly bound solutes from concanavalin A Sepharose Factors affecting the desorption of cottonmouth venom glycoproteins

    Journal:

    doi: 10.1016/j.chroma.2007.03.126

    Effects of 4 M urea on Con A Sepharose. A virgin Con A Sepharose column (column filters replaced) was subjected to a standard chromatographic experiment with 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside/4 M urea (pH 4.0) as the eluent.
    Figure Legend Snippet: Effects of 4 M urea on Con A Sepharose. A virgin Con A Sepharose column (column filters replaced) was subjected to a standard chromatographic experiment with 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside/4 M urea (pH 4.0) as the eluent.

    Techniques Used:

    Influence of pH on venom glycoprotein and PDE desorbed from Con A Sepharose. Eluents consisted of 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside titrated with NaOH to pH 3.0, 4.0, 5.0, or 6.0. Bradford data and PDE activities have been
    Figure Legend Snippet: Influence of pH on venom glycoprotein and PDE desorbed from Con A Sepharose. Eluents consisted of 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside titrated with NaOH to pH 3.0, 4.0, 5.0, or 6.0. Bradford data and PDE activities have been

    Techniques Used:

    Influence of auxiliary and alternative eluents on glycoprotein and PDE desorption from Con A Sepharose. Auxiliary eluents were added to the standard 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside (pH 4.0) buffer. These included 20% ethylene
    Figure Legend Snippet: Influence of auxiliary and alternative eluents on glycoprotein and PDE desorption from Con A Sepharose. Auxiliary eluents were added to the standard 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside (pH 4.0) buffer. These included 20% ethylene

    Techniques Used:

    Influence of eluent NaCl concentration on glycoprotein and PDE desorption. Increasing eluent NaCl concentrations reduces glycoprotein desorption from Con A Sepharose even after the Bradford and PDE assays are corrected for direct effects on the assay
    Figure Legend Snippet: Influence of eluent NaCl concentration on glycoprotein and PDE desorption. Increasing eluent NaCl concentrations reduces glycoprotein desorption from Con A Sepharose even after the Bradford and PDE assays are corrected for direct effects on the assay

    Techniques Used: Concentration Assay

    Glycoprotein elution profiles with 0-4 pauses introduced during desorption. The 1 mL Con A Sepharose column was equilibrated in 50 mM acetic acid/NaOH/500 mM NaCl/1 mM MnCl 2 /1 mM CaCl 2 /1 mM MgCl 2 (pH 6.0) and desorbed with 100 mM acetic acid/NaOH/1 M
    Figure Legend Snippet: Glycoprotein elution profiles with 0-4 pauses introduced during desorption. The 1 mL Con A Sepharose column was equilibrated in 50 mM acetic acid/NaOH/500 mM NaCl/1 mM MnCl 2 /1 mM CaCl 2 /1 mM MgCl 2 (pH 6.0) and desorbed with 100 mM acetic acid/NaOH/1 M

    Techniques Used:

    6) Product Images from "Elution of tightly bound solutes from concanavalin A Sepharose Factors affecting the desorption of cottonmouth venom glycoproteins"

    Article Title: Elution of tightly bound solutes from concanavalin A Sepharose Factors affecting the desorption of cottonmouth venom glycoproteins

    Journal:

    doi: 10.1016/j.chroma.2007.03.126

    Effects of 4 M urea on Con A Sepharose. A virgin Con A Sepharose column (column filters replaced) was subjected to a standard chromatographic experiment with 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside/4 M urea (pH 4.0) as the eluent.
    Figure Legend Snippet: Effects of 4 M urea on Con A Sepharose. A virgin Con A Sepharose column (column filters replaced) was subjected to a standard chromatographic experiment with 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside/4 M urea (pH 4.0) as the eluent.

    Techniques Used:

    Influence of pH on venom glycoprotein and PDE desorbed from Con A Sepharose. Eluents consisted of 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside titrated with NaOH to pH 3.0, 4.0, 5.0, or 6.0. Bradford data and PDE activities have been
    Figure Legend Snippet: Influence of pH on venom glycoprotein and PDE desorbed from Con A Sepharose. Eluents consisted of 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside titrated with NaOH to pH 3.0, 4.0, 5.0, or 6.0. Bradford data and PDE activities have been

    Techniques Used:

    Influence of auxiliary and alternative eluents on glycoprotein and PDE desorption from Con A Sepharose. Auxiliary eluents were added to the standard 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside (pH 4.0) buffer. These included 20% ethylene
    Figure Legend Snippet: Influence of auxiliary and alternative eluents on glycoprotein and PDE desorption from Con A Sepharose. Auxiliary eluents were added to the standard 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside (pH 4.0) buffer. These included 20% ethylene

    Techniques Used:

    Influence of eluent NaCl concentration on glycoprotein and PDE desorption. Increasing eluent NaCl concentrations reduces glycoprotein desorption from Con A Sepharose even after the Bradford and PDE assays are corrected for direct effects on the assay
    Figure Legend Snippet: Influence of eluent NaCl concentration on glycoprotein and PDE desorption. Increasing eluent NaCl concentrations reduces glycoprotein desorption from Con A Sepharose even after the Bradford and PDE assays are corrected for direct effects on the assay

    Techniques Used: Concentration Assay

    Glycoprotein elution profiles with 0-4 pauses introduced during desorption. The 1 mL Con A Sepharose column was equilibrated in 50 mM acetic acid/NaOH/500 mM NaCl/1 mM MnCl 2 /1 mM CaCl 2 /1 mM MgCl 2 (pH 6.0) and desorbed with 100 mM acetic acid/NaOH/1 M
    Figure Legend Snippet: Glycoprotein elution profiles with 0-4 pauses introduced during desorption. The 1 mL Con A Sepharose column was equilibrated in 50 mM acetic acid/NaOH/500 mM NaCl/1 mM MnCl 2 /1 mM CaCl 2 /1 mM MgCl 2 (pH 6.0) and desorbed with 100 mM acetic acid/NaOH/1 M

    Techniques Used:

    7) Product Images from "Elution of tightly bound solutes from concanavalin A Sepharose Factors affecting the desorption of cottonmouth venom glycoproteins"

    Article Title: Elution of tightly bound solutes from concanavalin A Sepharose Factors affecting the desorption of cottonmouth venom glycoproteins

    Journal:

    doi: 10.1016/j.chroma.2007.03.126

    Effects of 4 M urea on Con A Sepharose. A virgin Con A Sepharose column (column filters replaced) was subjected to a standard chromatographic experiment with 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside/4 M urea (pH 4.0) as the eluent.
    Figure Legend Snippet: Effects of 4 M urea on Con A Sepharose. A virgin Con A Sepharose column (column filters replaced) was subjected to a standard chromatographic experiment with 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside/4 M urea (pH 4.0) as the eluent.

    Techniques Used:

    Influence of pH on venom glycoprotein and PDE desorbed from Con A Sepharose. Eluents consisted of 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside titrated with NaOH to pH 3.0, 4.0, 5.0, or 6.0. Bradford data and PDE activities have been
    Figure Legend Snippet: Influence of pH on venom glycoprotein and PDE desorbed from Con A Sepharose. Eluents consisted of 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside titrated with NaOH to pH 3.0, 4.0, 5.0, or 6.0. Bradford data and PDE activities have been

    Techniques Used:

    Influence of auxiliary and alternative eluents on glycoprotein and PDE desorption from Con A Sepharose. Auxiliary eluents were added to the standard 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside (pH 4.0) buffer. These included 20% ethylene
    Figure Legend Snippet: Influence of auxiliary and alternative eluents on glycoprotein and PDE desorption from Con A Sepharose. Auxiliary eluents were added to the standard 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside (pH 4.0) buffer. These included 20% ethylene

    Techniques Used:

    Influence of eluent NaCl concentration on glycoprotein and PDE desorption. Increasing eluent NaCl concentrations reduces glycoprotein desorption from Con A Sepharose even after the Bradford and PDE assays are corrected for direct effects on the assay
    Figure Legend Snippet: Influence of eluent NaCl concentration on glycoprotein and PDE desorption. Increasing eluent NaCl concentrations reduces glycoprotein desorption from Con A Sepharose even after the Bradford and PDE assays are corrected for direct effects on the assay

    Techniques Used: Concentration Assay

    Glycoprotein elution profiles with 0-4 pauses introduced during desorption. The 1 mL Con A Sepharose column was equilibrated in 50 mM acetic acid/NaOH/500 mM NaCl/1 mM MnCl 2 /1 mM CaCl 2 /1 mM MgCl 2 (pH 6.0) and desorbed with 100 mM acetic acid/NaOH/1 M
    Figure Legend Snippet: Glycoprotein elution profiles with 0-4 pauses introduced during desorption. The 1 mL Con A Sepharose column was equilibrated in 50 mM acetic acid/NaOH/500 mM NaCl/1 mM MnCl 2 /1 mM CaCl 2 /1 mM MgCl 2 (pH 6.0) and desorbed with 100 mM acetic acid/NaOH/1 M

    Techniques Used:

    8) Product Images from "Saccharomyces cerevisiae CWH43"

    Article Title: Saccharomyces cerevisiae CWH43

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E07-05-0482

    Lipid remodeling of lipid moieties in GPI anchors to ceramides is defective in cwh43 Δ cells. (A) Analysis of PI moieties obtained from GPI-anchored proteins in wild-type (WT), per1 Δ, gup1 Δ, per1 Δ gup1 Δ, gpi7 Δ, and cwh43 Δ cells. Cells were labeled with [ 3 H]inositol at 30°C. Cell lysate was delipidated, and glycoproteins were concentrated using Con A-Sepharose. PI moieties of GPI anchors were released by deamination by using nitrous acid, separated by TLC by using solvent system 1, detected by autoradiography, and analyzed using a Molecular Imager FX. (B) Analysis of incorporation of [ 3 H]DHS derivatives in proteins. Cells were incubated for 20 min in the presence of myriocin and then labeled with [ 3 H]DHS at 30°C. Glycoproteins were concentrated from extracted lysate using Con A-Sepharose and separated by SDS-PAGE. Radiolabeled proteins were visualized using a Molecular Imager FX. Equal loading of total proteins was confirmed by Coomassie brilliant blue staining of the SDS-PAGE gels.
    Figure Legend Snippet: Lipid remodeling of lipid moieties in GPI anchors to ceramides is defective in cwh43 Δ cells. (A) Analysis of PI moieties obtained from GPI-anchored proteins in wild-type (WT), per1 Δ, gup1 Δ, per1 Δ gup1 Δ, gpi7 Δ, and cwh43 Δ cells. Cells were labeled with [ 3 H]inositol at 30°C. Cell lysate was delipidated, and glycoproteins were concentrated using Con A-Sepharose. PI moieties of GPI anchors were released by deamination by using nitrous acid, separated by TLC by using solvent system 1, detected by autoradiography, and analyzed using a Molecular Imager FX. (B) Analysis of incorporation of [ 3 H]DHS derivatives in proteins. Cells were incubated for 20 min in the presence of myriocin and then labeled with [ 3 H]DHS at 30°C. Glycoproteins were concentrated from extracted lysate using Con A-Sepharose and separated by SDS-PAGE. Radiolabeled proteins were visualized using a Molecular Imager FX. Equal loading of total proteins was confirmed by Coomassie brilliant blue staining of the SDS-PAGE gels.

    Techniques Used: Labeling, Thin Layer Chromatography, Autoradiography, Incubation, SDS Page, Staining

    9) Product Images from "Elution of tightly bound solutes from concanavalin A Sepharose Factors affecting the desorption of cottonmouth venom glycoproteins"

    Article Title: Elution of tightly bound solutes from concanavalin A Sepharose Factors affecting the desorption of cottonmouth venom glycoproteins

    Journal:

    doi: 10.1016/j.chroma.2007.03.126

    Effects of 4 M urea on Con A Sepharose. A virgin Con A Sepharose column (column filters replaced) was subjected to a standard chromatographic experiment with 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside/4 M urea (pH 4.0) as the eluent.
    Figure Legend Snippet: Effects of 4 M urea on Con A Sepharose. A virgin Con A Sepharose column (column filters replaced) was subjected to a standard chromatographic experiment with 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside/4 M urea (pH 4.0) as the eluent.

    Techniques Used:

    Influence of pH on venom glycoprotein and PDE desorbed from Con A Sepharose. Eluents consisted of 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside titrated with NaOH to pH 3.0, 4.0, 5.0, or 6.0. Bradford data and PDE activities have been
    Figure Legend Snippet: Influence of pH on venom glycoprotein and PDE desorbed from Con A Sepharose. Eluents consisted of 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside titrated with NaOH to pH 3.0, 4.0, 5.0, or 6.0. Bradford data and PDE activities have been

    Techniques Used:

    Influence of auxiliary and alternative eluents on glycoprotein and PDE desorption from Con A Sepharose. Auxiliary eluents were added to the standard 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside (pH 4.0) buffer. These included 20% ethylene
    Figure Legend Snippet: Influence of auxiliary and alternative eluents on glycoprotein and PDE desorption from Con A Sepharose. Auxiliary eluents were added to the standard 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside (pH 4.0) buffer. These included 20% ethylene

    Techniques Used:

    Influence of eluent NaCl concentration on glycoprotein and PDE desorption. Increasing eluent NaCl concentrations reduces glycoprotein desorption from Con A Sepharose even after the Bradford and PDE assays are corrected for direct effects on the assay
    Figure Legend Snippet: Influence of eluent NaCl concentration on glycoprotein and PDE desorption. Increasing eluent NaCl concentrations reduces glycoprotein desorption from Con A Sepharose even after the Bradford and PDE assays are corrected for direct effects on the assay

    Techniques Used: Concentration Assay

    Glycoprotein elution profiles with 0-4 pauses introduced during desorption. The 1 mL Con A Sepharose column was equilibrated in 50 mM acetic acid/NaOH/500 mM NaCl/1 mM MnCl 2 /1 mM CaCl 2 /1 mM MgCl 2 (pH 6.0) and desorbed with 100 mM acetic acid/NaOH/1 M
    Figure Legend Snippet: Glycoprotein elution profiles with 0-4 pauses introduced during desorption. The 1 mL Con A Sepharose column was equilibrated in 50 mM acetic acid/NaOH/500 mM NaCl/1 mM MnCl 2 /1 mM CaCl 2 /1 mM MgCl 2 (pH 6.0) and desorbed with 100 mM acetic acid/NaOH/1 M

    Techniques Used:

    10) Product Images from "Saccharomyces cerevisiae CWH43"

    Article Title: Saccharomyces cerevisiae CWH43

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E07-05-0482

    Lipid remodeling of lipid moieties in GPI anchors to ceramides is defective in cwh43 Δ cells. (A) Analysis of PI moieties obtained from GPI-anchored proteins in wild-type (WT), per1 Δ, gup1 Δ, per1 Δ gup1 Δ, gpi7 Δ, and cwh43 Δ cells. Cells were labeled with [ 3 H]inositol at 30°C. Cell lysate was delipidated, and glycoproteins were concentrated using Con A-Sepharose. PI moieties of GPI anchors were released by deamination by using nitrous acid, separated by TLC by using solvent system 1, detected by autoradiography, and analyzed using a Molecular Imager FX. (B) Analysis of incorporation of [ 3 H]DHS derivatives in proteins. Cells were incubated for 20 min in the presence of myriocin and then labeled with [ 3 H]DHS at 30°C. Glycoproteins were concentrated from extracted lysate using Con A-Sepharose and separated by SDS-PAGE. Radiolabeled proteins were visualized using a Molecular Imager FX. Equal loading of total proteins was confirmed by Coomassie brilliant blue staining of the SDS-PAGE gels.
    Figure Legend Snippet: Lipid remodeling of lipid moieties in GPI anchors to ceramides is defective in cwh43 Δ cells. (A) Analysis of PI moieties obtained from GPI-anchored proteins in wild-type (WT), per1 Δ, gup1 Δ, per1 Δ gup1 Δ, gpi7 Δ, and cwh43 Δ cells. Cells were labeled with [ 3 H]inositol at 30°C. Cell lysate was delipidated, and glycoproteins were concentrated using Con A-Sepharose. PI moieties of GPI anchors were released by deamination by using nitrous acid, separated by TLC by using solvent system 1, detected by autoradiography, and analyzed using a Molecular Imager FX. (B) Analysis of incorporation of [ 3 H]DHS derivatives in proteins. Cells were incubated for 20 min in the presence of myriocin and then labeled with [ 3 H]DHS at 30°C. Glycoproteins were concentrated from extracted lysate using Con A-Sepharose and separated by SDS-PAGE. Radiolabeled proteins were visualized using a Molecular Imager FX. Equal loading of total proteins was confirmed by Coomassie brilliant blue staining of the SDS-PAGE gels.

    Techniques Used: Labeling, Thin Layer Chromatography, Autoradiography, Incubation, SDS Page, Staining

    11) Product Images from "Saccharomyces cerevisiae CWH43"

    Article Title: Saccharomyces cerevisiae CWH43

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E07-05-0482

    Lipid remodeling of lipid moieties in GPI anchors to ceramides is defective in cwh43 Δ cells. (A) Analysis of PI moieties obtained from GPI-anchored proteins in wild-type (WT), per1 Δ, gup1 Δ, per1 Δ gup1 Δ, gpi7 Δ, and cwh43 Δ cells. Cells were labeled with [ 3 H]inositol at 30°C. Cell lysate was delipidated, and glycoproteins were concentrated using Con A-Sepharose. PI moieties of GPI anchors were released by deamination by using nitrous acid, separated by TLC by using solvent system 1, detected by autoradiography, and analyzed using a Molecular Imager FX. (B) Analysis of incorporation of [ 3 H]DHS derivatives in proteins. Cells were incubated for 20 min in the presence of myriocin and then labeled with [ 3 H]DHS at 30°C. Glycoproteins were concentrated from extracted lysate using Con A-Sepharose and separated by SDS-PAGE. Radiolabeled proteins were visualized using a Molecular Imager FX. Equal loading of total proteins was confirmed by Coomassie brilliant blue staining of the SDS-PAGE gels.
    Figure Legend Snippet: Lipid remodeling of lipid moieties in GPI anchors to ceramides is defective in cwh43 Δ cells. (A) Analysis of PI moieties obtained from GPI-anchored proteins in wild-type (WT), per1 Δ, gup1 Δ, per1 Δ gup1 Δ, gpi7 Δ, and cwh43 Δ cells. Cells were labeled with [ 3 H]inositol at 30°C. Cell lysate was delipidated, and glycoproteins were concentrated using Con A-Sepharose. PI moieties of GPI anchors were released by deamination by using nitrous acid, separated by TLC by using solvent system 1, detected by autoradiography, and analyzed using a Molecular Imager FX. (B) Analysis of incorporation of [ 3 H]DHS derivatives in proteins. Cells were incubated for 20 min in the presence of myriocin and then labeled with [ 3 H]DHS at 30°C. Glycoproteins were concentrated from extracted lysate using Con A-Sepharose and separated by SDS-PAGE. Radiolabeled proteins were visualized using a Molecular Imager FX. Equal loading of total proteins was confirmed by Coomassie brilliant blue staining of the SDS-PAGE gels.

    Techniques Used: Labeling, Thin Layer Chromatography, Autoradiography, Incubation, SDS Page, Staining

    12) Product Images from "Elution of tightly bound solutes from concanavalin A Sepharose Factors affecting the desorption of cottonmouth venom glycoproteins"

    Article Title: Elution of tightly bound solutes from concanavalin A Sepharose Factors affecting the desorption of cottonmouth venom glycoproteins

    Journal:

    doi: 10.1016/j.chroma.2007.03.126

    Effects of 4 M urea on Con A Sepharose. A virgin Con A Sepharose column (column filters replaced) was subjected to a standard chromatographic experiment with 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside/4 M urea (pH 4.0) as the eluent.
    Figure Legend Snippet: Effects of 4 M urea on Con A Sepharose. A virgin Con A Sepharose column (column filters replaced) was subjected to a standard chromatographic experiment with 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside/4 M urea (pH 4.0) as the eluent.

    Techniques Used:

    Influence of pH on venom glycoprotein and PDE desorbed from Con A Sepharose. Eluents consisted of 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside titrated with NaOH to pH 3.0, 4.0, 5.0, or 6.0. Bradford data and PDE activities have been
    Figure Legend Snippet: Influence of pH on venom glycoprotein and PDE desorbed from Con A Sepharose. Eluents consisted of 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside titrated with NaOH to pH 3.0, 4.0, 5.0, or 6.0. Bradford data and PDE activities have been

    Techniques Used:

    Influence of auxiliary and alternative eluents on glycoprotein and PDE desorption from Con A Sepharose. Auxiliary eluents were added to the standard 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside (pH 4.0) buffer. These included 20% ethylene
    Figure Legend Snippet: Influence of auxiliary and alternative eluents on glycoprotein and PDE desorption from Con A Sepharose. Auxiliary eluents were added to the standard 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside (pH 4.0) buffer. These included 20% ethylene

    Techniques Used:

    Influence of eluent NaCl concentration on glycoprotein and PDE desorption. Increasing eluent NaCl concentrations reduces glycoprotein desorption from Con A Sepharose even after the Bradford and PDE assays are corrected for direct effects on the assay
    Figure Legend Snippet: Influence of eluent NaCl concentration on glycoprotein and PDE desorption. Increasing eluent NaCl concentrations reduces glycoprotein desorption from Con A Sepharose even after the Bradford and PDE assays are corrected for direct effects on the assay

    Techniques Used: Concentration Assay

    Glycoprotein elution profiles with 0-4 pauses introduced during desorption. The 1 mL Con A Sepharose column was equilibrated in 50 mM acetic acid/NaOH/500 mM NaCl/1 mM MnCl 2 /1 mM CaCl 2 /1 mM MgCl 2 (pH 6.0) and desorbed with 100 mM acetic acid/NaOH/1 M
    Figure Legend Snippet: Glycoprotein elution profiles with 0-4 pauses introduced during desorption. The 1 mL Con A Sepharose column was equilibrated in 50 mM acetic acid/NaOH/500 mM NaCl/1 mM MnCl 2 /1 mM CaCl 2 /1 mM MgCl 2 (pH 6.0) and desorbed with 100 mM acetic acid/NaOH/1 M

    Techniques Used:

    13) Product Images from "Elution of tightly bound solutes from concanavalin A Sepharose Factors affecting the desorption of cottonmouth venom glycoproteins"

    Article Title: Elution of tightly bound solutes from concanavalin A Sepharose Factors affecting the desorption of cottonmouth venom glycoproteins

    Journal:

    doi: 10.1016/j.chroma.2007.03.126

    Effects of 4 M urea on Con A Sepharose. A virgin Con A Sepharose column (column filters replaced) was subjected to a standard chromatographic experiment with 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside/4 M urea (pH 4.0) as the eluent.
    Figure Legend Snippet: Effects of 4 M urea on Con A Sepharose. A virgin Con A Sepharose column (column filters replaced) was subjected to a standard chromatographic experiment with 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside/4 M urea (pH 4.0) as the eluent.

    Techniques Used:

    Influence of pH on venom glycoprotein and PDE desorbed from Con A Sepharose. Eluents consisted of 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside titrated with NaOH to pH 3.0, 4.0, 5.0, or 6.0. Bradford data and PDE activities have been
    Figure Legend Snippet: Influence of pH on venom glycoprotein and PDE desorbed from Con A Sepharose. Eluents consisted of 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside titrated with NaOH to pH 3.0, 4.0, 5.0, or 6.0. Bradford data and PDE activities have been

    Techniques Used:

    Influence of auxiliary and alternative eluents on glycoprotein and PDE desorption from Con A Sepharose. Auxiliary eluents were added to the standard 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside (pH 4.0) buffer. These included 20% ethylene
    Figure Legend Snippet: Influence of auxiliary and alternative eluents on glycoprotein and PDE desorption from Con A Sepharose. Auxiliary eluents were added to the standard 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside (pH 4.0) buffer. These included 20% ethylene

    Techniques Used:

    Influence of eluent NaCl concentration on glycoprotein and PDE desorption. Increasing eluent NaCl concentrations reduces glycoprotein desorption from Con A Sepharose even after the Bradford and PDE assays are corrected for direct effects on the assay
    Figure Legend Snippet: Influence of eluent NaCl concentration on glycoprotein and PDE desorption. Increasing eluent NaCl concentrations reduces glycoprotein desorption from Con A Sepharose even after the Bradford and PDE assays are corrected for direct effects on the assay

    Techniques Used: Concentration Assay

    Glycoprotein elution profiles with 0-4 pauses introduced during desorption. The 1 mL Con A Sepharose column was equilibrated in 50 mM acetic acid/NaOH/500 mM NaCl/1 mM MnCl 2 /1 mM CaCl 2 /1 mM MgCl 2 (pH 6.0) and desorbed with 100 mM acetic acid/NaOH/1 M
    Figure Legend Snippet: Glycoprotein elution profiles with 0-4 pauses introduced during desorption. The 1 mL Con A Sepharose column was equilibrated in 50 mM acetic acid/NaOH/500 mM NaCl/1 mM MnCl 2 /1 mM CaCl 2 /1 mM MgCl 2 (pH 6.0) and desorbed with 100 mM acetic acid/NaOH/1 M

    Techniques Used:

    14) Product Images from "Elution of tightly bound solutes from concanavalin A Sepharose Factors affecting the desorption of cottonmouth venom glycoproteins"

    Article Title: Elution of tightly bound solutes from concanavalin A Sepharose Factors affecting the desorption of cottonmouth venom glycoproteins

    Journal:

    doi: 10.1016/j.chroma.2007.03.126

    Effects of 4 M urea on Con A Sepharose. A virgin Con A Sepharose column (column filters replaced) was subjected to a standard chromatographic experiment with 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside/4 M urea (pH 4.0) as the eluent.
    Figure Legend Snippet: Effects of 4 M urea on Con A Sepharose. A virgin Con A Sepharose column (column filters replaced) was subjected to a standard chromatographic experiment with 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside/4 M urea (pH 4.0) as the eluent.

    Techniques Used:

    Influence of pH on venom glycoprotein and PDE desorbed from Con A Sepharose. Eluents consisted of 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside titrated with NaOH to pH 3.0, 4.0, 5.0, or 6.0. Bradford data and PDE activities have been
    Figure Legend Snippet: Influence of pH on venom glycoprotein and PDE desorbed from Con A Sepharose. Eluents consisted of 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside titrated with NaOH to pH 3.0, 4.0, 5.0, or 6.0. Bradford data and PDE activities have been

    Techniques Used:

    Influence of auxiliary and alternative eluents on glycoprotein and PDE desorption from Con A Sepharose. Auxiliary eluents were added to the standard 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside (pH 4.0) buffer. These included 20% ethylene
    Figure Legend Snippet: Influence of auxiliary and alternative eluents on glycoprotein and PDE desorption from Con A Sepharose. Auxiliary eluents were added to the standard 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside (pH 4.0) buffer. These included 20% ethylene

    Techniques Used:

    Influence of eluent NaCl concentration on glycoprotein and PDE desorption. Increasing eluent NaCl concentrations reduces glycoprotein desorption from Con A Sepharose even after the Bradford and PDE assays are corrected for direct effects on the assay
    Figure Legend Snippet: Influence of eluent NaCl concentration on glycoprotein and PDE desorption. Increasing eluent NaCl concentrations reduces glycoprotein desorption from Con A Sepharose even after the Bradford and PDE assays are corrected for direct effects on the assay

    Techniques Used: Concentration Assay

    Glycoprotein elution profiles with 0-4 pauses introduced during desorption. The 1 mL Con A Sepharose column was equilibrated in 50 mM acetic acid/NaOH/500 mM NaCl/1 mM MnCl 2 /1 mM CaCl 2 /1 mM MgCl 2 (pH 6.0) and desorbed with 100 mM acetic acid/NaOH/1 M
    Figure Legend Snippet: Glycoprotein elution profiles with 0-4 pauses introduced during desorption. The 1 mL Con A Sepharose column was equilibrated in 50 mM acetic acid/NaOH/500 mM NaCl/1 mM MnCl 2 /1 mM CaCl 2 /1 mM MgCl 2 (pH 6.0) and desorbed with 100 mM acetic acid/NaOH/1 M

    Techniques Used:

    Related Articles

    other:

    Article Title: Elution of tightly bound solutes from concanavalin A Sepharose Factors affecting the desorption of cottonmouth venom glycoproteins
    Article Snippet: Having said this, it must be added that Con A Sepharose is a remarkably resilient resin.

    Article Title: Combining Lectin Microcolumns with High-Resolution Separation Techniques for Enrichment of Glycoproteins and Glycopeptides
    Article Snippet: Con A Sepharose was acquired from Amersham Biosciences AB (Uppsala, Sweden).

    Article Title: Elution of tightly bound solutes from concanavalin A Sepharose Factors affecting the desorption of cottonmouth venom glycoproteins
    Article Snippet: Wan and Hasenstein [ ] reported no complications when applying samples containing 4 M urea to Con A Sepharose.

    Article Title: Elution of tightly bound solutes from concanavalin A Sepharose Factors affecting the desorption of cottonmouth venom glycoproteins
    Article Snippet: Counterintuitively, higher pyranoside concentrations actually diminished the amount of venom glycoprotein desorbed from Con A Sepharose, regardless of which pyranoside was used ( ).

    Activated Clotting Time Assay:

    Article Title: Elution of tightly bound solutes from concanavalin A Sepharose Factors affecting the desorption of cottonmouth venom glycoproteins
    Article Snippet: .. According to the instruction sheet for Con A Sepharose, “Borate is known to form complexes with cis-diols on sugar residues and thus act as an competing eluent. .. For elution with borate, use a 0.1 M borate buffer, pH 6.5.” The recommended borate buffer desorbed only ∼10% as much glycoprotein as the standard buffer system (two-tailed p < 0.0001, Mann-Whitney test) ( ).

    Clarification Assay:

    Article Title: Molecular and antigenic characterization of Trypanosoma cruzi TolT proteins
    Article Snippet: Concanavalin A fractionation and endoglycosidase H treatment Trypomastigote pellets (3 x 108 ) were homogenized in 500 μL of ConA buffer [50 mM Tris/HCl, pH 7.4, 150 mM NaCl, 1% (v/v) Triton X-100, 0.1% (v/v) Nonidet P40, 0.1% (w/v) sodium deoxycholate, 5 mM Cl2 Ca, 5 mM Cl2 Mg, 5 mM Cl2 Mn, 1% (v/v) inhibitor protease cocktail (Sigma), and 1 mM DTT] and processed as described [ ]. .. After clarification, parasite extract was fractionated overnight at 4°C onto 100 μL of ConA–Sepharose (GE Healthcare), and retained glycoproteins eluted with 50 μL of SDS-PAGE loading buffer. .. Flow-through and ConA-bound fractions were analyzed by Western blot with different antisera.

    SDS Page:

    Article Title: Molecular and antigenic characterization of Trypanosoma cruzi TolT proteins
    Article Snippet: Concanavalin A fractionation and endoglycosidase H treatment Trypomastigote pellets (3 x 108 ) were homogenized in 500 μL of ConA buffer [50 mM Tris/HCl, pH 7.4, 150 mM NaCl, 1% (v/v) Triton X-100, 0.1% (v/v) Nonidet P40, 0.1% (w/v) sodium deoxycholate, 5 mM Cl2 Ca, 5 mM Cl2 Mg, 5 mM Cl2 Mn, 1% (v/v) inhibitor protease cocktail (Sigma), and 1 mM DTT] and processed as described [ ]. .. After clarification, parasite extract was fractionated overnight at 4°C onto 100 μL of ConA–Sepharose (GE Healthcare), and retained glycoproteins eluted with 50 μL of SDS-PAGE loading buffer. .. Flow-through and ConA-bound fractions were analyzed by Western blot with different antisera.

    Concentration Assay:

    Article Title: Elution of tightly bound solutes from concanavalin A Sepharose Factors affecting the desorption of cottonmouth venom glycoproteins
    Article Snippet: .. The use of 4-6 pauses of 5-10 min duration during solute desorption greatly improves glycoprotein yields from Con A Sepharose without increasing the eluent volume or the concentration of desorbent used. .. With hydrophobic glycoproteins, NaCl and other salts enhance binding and should be avoided in elution buffers.

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  • 93
    GE Healthcare pre packed heparin sepharose resin
    Serp-1 binds to heparin in vitro. In a pull-down experiment with <t>heparin/sepharose</t> beads (A) , Serp-1 (100 ng) was incubated with 15 µl of heparin/sepharose beads (Amersham) for one hour at 37 ° C. After Serp-1 bound to heparin beads, 100 µl of NaCl solution at different concentrations (0 M, 1 M, and 2 M) was added to elute Serp-1 from the heparin beads. The elution was performed at room temperature for 10 minutes. After elution, the Serp-1 heparin beads were loaded on SDS-PAGE gel for electrophoresis and followed by the immunoblot detection with the anti-Serp-1 monoclonal antibody. 100 ng Serp-1 positive control shown in lane 1. FPLC chromatograph analysis: native (B) and heat-inactivated (C) Serp-1 were loaded onto the heparin columns and eluted with a NaCl gradient. The absorbance at 280 nm was monitored. A representative elution curve is presented from three repeat experiments. X axis at bottom indicates running time of the column analysis. X axis at top indicates collecting fractions (1 ml/fraction). Y axis indicates absorbance at 280 nm. Amino acid sequence alignment (D). Human PAI-1 helix D heparin-binding domain (13 amino acid residues) aligns with the Serp-1 sequence by MacVector (version 3). The residue (Arg 6 ) in Serp-1 has been mutated (boxed) to examine its significance of the heparin binding ability (see text). The identical amino acids in both sequences are dotted and the similar amino acids are starred underneath. 3-D serpin structure showing the relative positions between helix D (hD) and the reactive center loop (RCL) is adapted from Gettins (Chem Rev 2002)
    Pre Packed Heparin Sepharose Resin, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    GE Healthcare q sepharose anion exchange
    The AND-1 CT interacts with the N-terminal domain of the B subunit. ( a ) Pulldown experiment on glutathione <t>agarose</t> of full-length AND-1 with different GST constructs of the B subunit, analysed by SDS–PAGE and Coomassie staining. The domain structure of the B subunit is shown above the experiment, together with the PDB ID of each domain. The boundaries of each B construct tested in the pulldown are also shown. ( b ) Pulldown experiment on glutathione agarose of AND-1 constructs with GST-B NTD (amino acids 1–78), analysed by SDS–PAGE and Coomassie staining. The domain structure of human AND-1 is shown above the experiment, together with the PDB ID of each domain. The boundaries of each AND-1 construct tested in the pulldown experiment are also shown.
    Q Sepharose Anion Exchange, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    GE Healthcare biotin labeled anti pgc 1α
    Tug1 and <t>PGC-1α</t> directly interact to enhance Ppargc1a mRNA levels. ( A ) Proposed model for the Tug1 mediated enhancement of Ppargc1a transcription. ( B ) Prediction of interaction propensity between the CTD and full length Tug1 RNA. Positive interaction score predicts increased propensity of binding. Control interaction plot (bottom panel) depicts experimentally validated interaction between the lncRNA Xist and its binding partner SRSF1. Positive Interaction Score indicates predicted binding. ( C ) Western blot (WB) analysis of podocyte nuclear extract incubated with sense or antisense biotinylated Tug1 demonstrating PGC-1α interaction with sense Tug1 RNA. ( D ) Western blot analysis confirming IP of endogenous PGC-1α from podocytes. qPCR analysis for Tug1 RNA in PGC-1α or IgG immunoprecipitated extracts. ( E ) qPCR analysis of RNA isolated from IP with Flag antibody from HEK293T cells transfected with Flag-tagged WT or the CTD deletion mutant (ΔCTD) PGC-1α. ( F ) Diagram of the GST PGC-1α variants used in domain mapping. ( G ) qPCR analysis of in vitro binding reactions using in vitro–transcribed Tug1 RNA or a luciferase RNA control incubated with the fusion protein constructs described in F . Agarose gel of qPCR products of Tug1 and luciferase. ( H ) Left: ChIP-qPCR analysis of PGC-1α in control WT (Ctrl) and Tug1 -KD cells at the upstream TBE element, an intergenic region devoid of the predicted PGC-1α interaction, and the Ppargc1a gene promoter (primers spanning the distal, medial, and proximal regions of the promoter). Right: Positive control ChIP with primers designed to detect the promoter region of Pdk4 . Data were fold-enrichment normalized to IgG. Cell culture experiments were repeated at least 3 times. *** P
    Biotin Labeled Anti Pgc 1α, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    GE Healthcare ns1 rna
    ( A ) The number of viral transcripts of <t>NS1</t> compared to VP1/2. ( B ) Viral DNA loads in EMBs with active or latent infection. ( C ) Viral DNA load compared between EMBs with detectable <t>VP1/2-RNA,</t> NS1-RNA or NS1- and VP1/2-RNA expression (ANOVA p = 0.0427). Numbers above the bars represent p-values.
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    Serp-1 binds to heparin in vitro. In a pull-down experiment with heparin/sepharose beads (A) , Serp-1 (100 ng) was incubated with 15 µl of heparin/sepharose beads (Amersham) for one hour at 37 ° C. After Serp-1 bound to heparin beads, 100 µl of NaCl solution at different concentrations (0 M, 1 M, and 2 M) was added to elute Serp-1 from the heparin beads. The elution was performed at room temperature for 10 minutes. After elution, the Serp-1 heparin beads were loaded on SDS-PAGE gel for electrophoresis and followed by the immunoblot detection with the anti-Serp-1 monoclonal antibody. 100 ng Serp-1 positive control shown in lane 1. FPLC chromatograph analysis: native (B) and heat-inactivated (C) Serp-1 were loaded onto the heparin columns and eluted with a NaCl gradient. The absorbance at 280 nm was monitored. A representative elution curve is presented from three repeat experiments. X axis at bottom indicates running time of the column analysis. X axis at top indicates collecting fractions (1 ml/fraction). Y axis indicates absorbance at 280 nm. Amino acid sequence alignment (D). Human PAI-1 helix D heparin-binding domain (13 amino acid residues) aligns with the Serp-1 sequence by MacVector (version 3). The residue (Arg 6 ) in Serp-1 has been mutated (boxed) to examine its significance of the heparin binding ability (see text). The identical amino acids in both sequences are dotted and the similar amino acids are starred underneath. 3-D serpin structure showing the relative positions between helix D (hD) and the reactive center loop (RCL) is adapted from Gettins (Chem Rev 2002)

    Journal: The Open Biochemistry Journal

    Article Title: Heparin Alters Viral Serpin, Serp-1, Anti-Thrombolytic Activity to Anti-Thrombotic Activity

    doi: 10.2174/1874091X00802010006

    Figure Lengend Snippet: Serp-1 binds to heparin in vitro. In a pull-down experiment with heparin/sepharose beads (A) , Serp-1 (100 ng) was incubated with 15 µl of heparin/sepharose beads (Amersham) for one hour at 37 ° C. After Serp-1 bound to heparin beads, 100 µl of NaCl solution at different concentrations (0 M, 1 M, and 2 M) was added to elute Serp-1 from the heparin beads. The elution was performed at room temperature for 10 minutes. After elution, the Serp-1 heparin beads were loaded on SDS-PAGE gel for electrophoresis and followed by the immunoblot detection with the anti-Serp-1 monoclonal antibody. 100 ng Serp-1 positive control shown in lane 1. FPLC chromatograph analysis: native (B) and heat-inactivated (C) Serp-1 were loaded onto the heparin columns and eluted with a NaCl gradient. The absorbance at 280 nm was monitored. A representative elution curve is presented from three repeat experiments. X axis at bottom indicates running time of the column analysis. X axis at top indicates collecting fractions (1 ml/fraction). Y axis indicates absorbance at 280 nm. Amino acid sequence alignment (D). Human PAI-1 helix D heparin-binding domain (13 amino acid residues) aligns with the Serp-1 sequence by MacVector (version 3). The residue (Arg 6 ) in Serp-1 has been mutated (boxed) to examine its significance of the heparin binding ability (see text). The identical amino acids in both sequences are dotted and the similar amino acids are starred underneath. 3-D serpin structure showing the relative positions between helix D (hD) and the reactive center loop (RCL) is adapted from Gettins (Chem Rev 2002)

    Article Snippet: Heparin / Sepharose Column Chromatography Heparin binding affinity analysis was performed using a fast protein liquid chromatography (FPLC) system (Pharmacia LKB Biotechnology Inc.) and a 1-ml column of pre-packed Heparin/Sepharose resin (HiTrap, Amersham).

    Techniques: In Vitro, Incubation, SDS Page, Electrophoresis, Positive Control, Fast Protein Liquid Chromatography, Sequencing, Binding Assay

    The AND-1 CT interacts with the N-terminal domain of the B subunit. ( a ) Pulldown experiment on glutathione agarose of full-length AND-1 with different GST constructs of the B subunit, analysed by SDS–PAGE and Coomassie staining. The domain structure of the B subunit is shown above the experiment, together with the PDB ID of each domain. The boundaries of each B construct tested in the pulldown are also shown. ( b ) Pulldown experiment on glutathione agarose of AND-1 constructs with GST-B NTD (amino acids 1–78), analysed by SDS–PAGE and Coomassie staining. The domain structure of human AND-1 is shown above the experiment, together with the PDB ID of each domain. The boundaries of each AND-1 construct tested in the pulldown experiment are also shown.

    Journal: Open Biology

    Article Title: The human CTF4-orthologue AND-1 interacts with DNA polymerase α/primase via its unique C-terminal HMG box

    doi: 10.1098/rsob.170217

    Figure Lengend Snippet: The AND-1 CT interacts with the N-terminal domain of the B subunit. ( a ) Pulldown experiment on glutathione agarose of full-length AND-1 with different GST constructs of the B subunit, analysed by SDS–PAGE and Coomassie staining. The domain structure of the B subunit is shown above the experiment, together with the PDB ID of each domain. The boundaries of each B construct tested in the pulldown are also shown. ( b ) Pulldown experiment on glutathione agarose of AND-1 constructs with GST-B NTD (amino acids 1–78), analysed by SDS–PAGE and Coomassie staining. The domain structure of human AND-1 is shown above the experiment, together with the PDB ID of each domain. The boundaries of each AND-1 construct tested in the pulldown experiment are also shown.

    Article Snippet: Purification involved successive Ni-NTA agarose (Qiagen) and Q-Sepharose anion-exchange (GE Healthcare) chromatography steps.

    Techniques: Construct, SDS Page, Staining

    The interaction of AND-1 with the B subunit is stronger than the interaction with Pol α CIP. ( a ) Full-length His-myc-AND-1 pulldown by equimolar amounts of GST-tagged Pol α CIP and B NTD on glutathione agarose. GST-CIP AA was used as negative control ( figure 2 d ). (Top panel) Coomassie-stained SDS–PAGE gel. (Bottom panel) Corresponding anti-His western blot (WB). ( b ) Pulldown of WT and M1051E AND-1 by Pol α/primase. StrepII-tagged Pol α/primase was immobilized on Strep-Tactin Superflow resin and released by desthiobiotin. (Top panel) Coomassie-stained SDS–PAGE. (Bottom panel) Corresponding anti-His western blot. ( c ) Competition co-precipitation. Full-length StrepII-tagged Pol α/primase was tested for interaction with full-length His-myc-AND-1 on Strep-Tactin Superflow resin, in the presence of excess purified B NTD or Pol α CIP. AND-1 control comprised resin only with AND-1. (Top panel) Coomassie-stained SDS–PAGE. (Bottom panel) corresponding anti-His western blot (His-tagged PriS is also detected).

    Journal: Open Biology

    Article Title: The human CTF4-orthologue AND-1 interacts with DNA polymerase α/primase via its unique C-terminal HMG box

    doi: 10.1098/rsob.170217

    Figure Lengend Snippet: The interaction of AND-1 with the B subunit is stronger than the interaction with Pol α CIP. ( a ) Full-length His-myc-AND-1 pulldown by equimolar amounts of GST-tagged Pol α CIP and B NTD on glutathione agarose. GST-CIP AA was used as negative control ( figure 2 d ). (Top panel) Coomassie-stained SDS–PAGE gel. (Bottom panel) Corresponding anti-His western blot (WB). ( b ) Pulldown of WT and M1051E AND-1 by Pol α/primase. StrepII-tagged Pol α/primase was immobilized on Strep-Tactin Superflow resin and released by desthiobiotin. (Top panel) Coomassie-stained SDS–PAGE. (Bottom panel) Corresponding anti-His western blot. ( c ) Competition co-precipitation. Full-length StrepII-tagged Pol α/primase was tested for interaction with full-length His-myc-AND-1 on Strep-Tactin Superflow resin, in the presence of excess purified B NTD or Pol α CIP. AND-1 control comprised resin only with AND-1. (Top panel) Coomassie-stained SDS–PAGE. (Bottom panel) corresponding anti-His western blot (His-tagged PriS is also detected).

    Article Snippet: Purification involved successive Ni-NTA agarose (Qiagen) and Q-Sepharose anion-exchange (GE Healthcare) chromatography steps.

    Techniques: Negative Control, Staining, SDS Page, Western Blot, Purification

    Tug1 and PGC-1α directly interact to enhance Ppargc1a mRNA levels. ( A ) Proposed model for the Tug1 mediated enhancement of Ppargc1a transcription. ( B ) Prediction of interaction propensity between the CTD and full length Tug1 RNA. Positive interaction score predicts increased propensity of binding. Control interaction plot (bottom panel) depicts experimentally validated interaction between the lncRNA Xist and its binding partner SRSF1. Positive Interaction Score indicates predicted binding. ( C ) Western blot (WB) analysis of podocyte nuclear extract incubated with sense or antisense biotinylated Tug1 demonstrating PGC-1α interaction with sense Tug1 RNA. ( D ) Western blot analysis confirming IP of endogenous PGC-1α from podocytes. qPCR analysis for Tug1 RNA in PGC-1α or IgG immunoprecipitated extracts. ( E ) qPCR analysis of RNA isolated from IP with Flag antibody from HEK293T cells transfected with Flag-tagged WT or the CTD deletion mutant (ΔCTD) PGC-1α. ( F ) Diagram of the GST PGC-1α variants used in domain mapping. ( G ) qPCR analysis of in vitro binding reactions using in vitro–transcribed Tug1 RNA or a luciferase RNA control incubated with the fusion protein constructs described in F . Agarose gel of qPCR products of Tug1 and luciferase. ( H ) Left: ChIP-qPCR analysis of PGC-1α in control WT (Ctrl) and Tug1 -KD cells at the upstream TBE element, an intergenic region devoid of the predicted PGC-1α interaction, and the Ppargc1a gene promoter (primers spanning the distal, medial, and proximal regions of the promoter). Right: Positive control ChIP with primers designed to detect the promoter region of Pdk4 . Data were fold-enrichment normalized to IgG. Cell culture experiments were repeated at least 3 times. *** P

    Journal: The Journal of Clinical Investigation

    Article Title: Long noncoding RNA Tug1 regulates mitochondrial bioenergetics in diabetic nephropathy

    doi: 10.1172/JCI87927

    Figure Lengend Snippet: Tug1 and PGC-1α directly interact to enhance Ppargc1a mRNA levels. ( A ) Proposed model for the Tug1 mediated enhancement of Ppargc1a transcription. ( B ) Prediction of interaction propensity between the CTD and full length Tug1 RNA. Positive interaction score predicts increased propensity of binding. Control interaction plot (bottom panel) depicts experimentally validated interaction between the lncRNA Xist and its binding partner SRSF1. Positive Interaction Score indicates predicted binding. ( C ) Western blot (WB) analysis of podocyte nuclear extract incubated with sense or antisense biotinylated Tug1 demonstrating PGC-1α interaction with sense Tug1 RNA. ( D ) Western blot analysis confirming IP of endogenous PGC-1α from podocytes. qPCR analysis for Tug1 RNA in PGC-1α or IgG immunoprecipitated extracts. ( E ) qPCR analysis of RNA isolated from IP with Flag antibody from HEK293T cells transfected with Flag-tagged WT or the CTD deletion mutant (ΔCTD) PGC-1α. ( F ) Diagram of the GST PGC-1α variants used in domain mapping. ( G ) qPCR analysis of in vitro binding reactions using in vitro–transcribed Tug1 RNA or a luciferase RNA control incubated with the fusion protein constructs described in F . Agarose gel of qPCR products of Tug1 and luciferase. ( H ) Left: ChIP-qPCR analysis of PGC-1α in control WT (Ctrl) and Tug1 -KD cells at the upstream TBE element, an intergenic region devoid of the predicted PGC-1α interaction, and the Ppargc1a gene promoter (primers spanning the distal, medial, and proximal regions of the promoter). Right: Positive control ChIP with primers designed to detect the promoter region of Pdk4 . Data were fold-enrichment normalized to IgG. Cell culture experiments were repeated at least 3 times. *** P

    Article Snippet: For endogenous RIP assay using antibodies, biotin-labeled anti–PGC-1α (NBP1-04676B; Novus) or normal rabbit IgG (sc-2027; Santa Cruz Biotechnology Inc.) was incubated with total lysate at 4ºC overnight, then Streptavidin Dynabeads M-280 (Thermo Fischer Scientific) were added and incubated for 2 hours.

    Techniques: Pyrolysis Gas Chromatography, Binding Assay, Western Blot, Incubation, Real-time Polymerase Chain Reaction, Immunoprecipitation, Isolation, Transfection, Mutagenesis, In Vitro, Luciferase, Construct, Agarose Gel Electrophoresis, Chromatin Immunoprecipitation, Positive Control, Cell Culture

    Tug1 mediates expression of PGC-1α pathway genes. ( A ) Gene expression analysis of RNA from pGIPZ-shControl (shCtrl) or sh Tug1 lentivirus–transduced podocytes used for microarray analysis. ( B ) Volcano plot of microarray data generated from Tug1 -KD podocytes compared with controls. A cutoff of a log 2 fold-change greater than 2 and a –log 10 ( P value) greater than 1 was used for downstream pathway analysis. ( C – E ) Bioinformatics analysis of differentially regulated Tug1 target genes. ( C and D ) Biological processes GO terms from genes differentially up- and downregulated by Tug1 . ( E ) Pathway analysis of Tug1 -downregulated genes. ( F ) Hierarchical clustering analysis of RNA expression levels of PGC-1α–related genes in control podocytes compared with podocytes harboring stable KD of Tug1 . Yellow boxes highlight genes that are direct targets of PGC-1α, and red boxes highlight its upstream regulators. ( G ) qPCR validation of several direct targets of PGC-1α. Expression values were normalized to Gapdh internal controls. Cell culture experiments were repeated at least 3 times. *P

    Journal: The Journal of Clinical Investigation

    Article Title: Long noncoding RNA Tug1 regulates mitochondrial bioenergetics in diabetic nephropathy

    doi: 10.1172/JCI87927

    Figure Lengend Snippet: Tug1 mediates expression of PGC-1α pathway genes. ( A ) Gene expression analysis of RNA from pGIPZ-shControl (shCtrl) or sh Tug1 lentivirus–transduced podocytes used for microarray analysis. ( B ) Volcano plot of microarray data generated from Tug1 -KD podocytes compared with controls. A cutoff of a log 2 fold-change greater than 2 and a –log 10 ( P value) greater than 1 was used for downstream pathway analysis. ( C – E ) Bioinformatics analysis of differentially regulated Tug1 target genes. ( C and D ) Biological processes GO terms from genes differentially up- and downregulated by Tug1 . ( E ) Pathway analysis of Tug1 -downregulated genes. ( F ) Hierarchical clustering analysis of RNA expression levels of PGC-1α–related genes in control podocytes compared with podocytes harboring stable KD of Tug1 . Yellow boxes highlight genes that are direct targets of PGC-1α, and red boxes highlight its upstream regulators. ( G ) qPCR validation of several direct targets of PGC-1α. Expression values were normalized to Gapdh internal controls. Cell culture experiments were repeated at least 3 times. *P

    Article Snippet: For endogenous RIP assay using antibodies, biotin-labeled anti–PGC-1α (NBP1-04676B; Novus) or normal rabbit IgG (sc-2027; Santa Cruz Biotechnology Inc.) was incubated with total lysate at 4ºC overnight, then Streptavidin Dynabeads M-280 (Thermo Fischer Scientific) were added and incubated for 2 hours.

    Techniques: Expressing, Pyrolysis Gas Chromatography, Microarray, Generated, RNA Expression, Real-time Polymerase Chain Reaction, Cell Culture

    Tug1 modulates Ppargc1a mRNA and mitochondrial bioenergetics in vitro. ( A and B ) qPCR analysis of Ppargc1a and Tug1 in control (Lenti ctrl), Tug1 -KD, or Tug1 -OE cultured podocytes under NG or HG conditions. ( C ) qPCR of selected PGC-1α direct targets in podocytes from A . ( D ) Measurement of mitochondrial ROS in cultured podocytes, measured by flow cytometry as the MFI of MitoSOX. ( E ) Flow cytometric determination of the percentage of podocytes that underwent apoptosis as measured by annexin V–FITC + cells. ( F and G ) Measurement of complex I and III activity in mitochondria isolated from cultured podocytes. ( H ) Relative ATP levels in podocytes, normalized to total protein content. ( I – K ) Mitochondrial OCR analysis of cultured podocytes using the Seahorse XF24 Bioanalyzer device. Oligomycin (2 μM), FCCP (2 μM), and rotenone (0.5 μM)/antimycin A (0.5 μM) were added at the times indicated by dashed lines. Basal and maximal respiratory rates of podocytes were determined by calculating the AUC. Cell culture experiments were repeated at least 3 times. * P

    Journal: The Journal of Clinical Investigation

    Article Title: Long noncoding RNA Tug1 regulates mitochondrial bioenergetics in diabetic nephropathy

    doi: 10.1172/JCI87927

    Figure Lengend Snippet: Tug1 modulates Ppargc1a mRNA and mitochondrial bioenergetics in vitro. ( A and B ) qPCR analysis of Ppargc1a and Tug1 in control (Lenti ctrl), Tug1 -KD, or Tug1 -OE cultured podocytes under NG or HG conditions. ( C ) qPCR of selected PGC-1α direct targets in podocytes from A . ( D ) Measurement of mitochondrial ROS in cultured podocytes, measured by flow cytometry as the MFI of MitoSOX. ( E ) Flow cytometric determination of the percentage of podocytes that underwent apoptosis as measured by annexin V–FITC + cells. ( F and G ) Measurement of complex I and III activity in mitochondria isolated from cultured podocytes. ( H ) Relative ATP levels in podocytes, normalized to total protein content. ( I – K ) Mitochondrial OCR analysis of cultured podocytes using the Seahorse XF24 Bioanalyzer device. Oligomycin (2 μM), FCCP (2 μM), and rotenone (0.5 μM)/antimycin A (0.5 μM) were added at the times indicated by dashed lines. Basal and maximal respiratory rates of podocytes were determined by calculating the AUC. Cell culture experiments were repeated at least 3 times. * P

    Article Snippet: For endogenous RIP assay using antibodies, biotin-labeled anti–PGC-1α (NBP1-04676B; Novus) or normal rabbit IgG (sc-2027; Santa Cruz Biotechnology Inc.) was incubated with total lysate at 4ºC overnight, then Streptavidin Dynabeads M-280 (Thermo Fischer Scientific) were added and incubated for 2 hours.

    Techniques: In Vitro, Real-time Polymerase Chain Reaction, Cell Culture, Pyrolysis Gas Chromatography, Flow Cytometry, Cytometry, Activity Assay, Isolation

    ( A ) The number of viral transcripts of NS1 compared to VP1/2. ( B ) Viral DNA loads in EMBs with active or latent infection. ( C ) Viral DNA load compared between EMBs with detectable VP1/2-RNA, NS1-RNA or NS1- and VP1/2-RNA expression (ANOVA p = 0.0427). Numbers above the bars represent p-values.

    Journal: Scientific Reports

    Article Title: Detection of parvovirus mRNAs as markers for viral activity in endomyocardial biopsy-based diagnosis of patients with unexplained heart failure

    doi: 10.1038/s41598-020-78597-4

    Figure Lengend Snippet: ( A ) The number of viral transcripts of NS1 compared to VP1/2. ( B ) Viral DNA loads in EMBs with active or latent infection. ( C ) Viral DNA load compared between EMBs with detectable VP1/2-RNA, NS1-RNA or NS1- and VP1/2-RNA expression (ANOVA p = 0.0427). Numbers above the bars represent p-values.

    Article Snippet: When both transcription intermediates (5156 ± 1076 GE/µg) or only VP1/2-RNA (5577 ± 1786 GE/µg) were detectable, the load of viral genomes was significantly increased compared to EMBs of patients that expressed solely NS1 RNA (2863 ± 454.2 GE/µg) (p = 0.0237; p = 0.0354) (Fig. C) Overall difference of viral load between these three groups was statistically significant (ANOVA p = 0.0427).

    Techniques: Infection, RNA Expression

    ( A ) Schematic description of the B19V genome organization and PCR design. Gene locus and primer and probe localization for NS1 and VP1/2 are indicated by numbers representing the nucleotide position. ITR inverted terminal repeat, NS1 non structural protein 1, VP1/2 capsid proteins, P6 P6 promotor, (pA)p polyadenylation site proximal, (pA)d polyadenylation site distal. ( B ) Representative agarose gel electrophoresis gel blot image of a B19V-VP1/2 DNA and RNA-positive EMB using VP1/2 specific nested-PCR. Amplicon length 173 bps. 1 = DNA-Marker 100 bps; 2 = positive control; 3 = negative control; 4 = PCR after DNA extraction and DNAse treatment; 5 = PCR after RNA extraction, RNAse treatment and RT-PCR; 6 = PCR after DNA extraction; 7 = PCR after RNA extraction and DNAse treatment and RT-PCR. Complete gel blot image of figure ( B ) was shown in Supplementary Fig. S1 . ( C ) Representative agarose gel electrophoresis gel blot image of 10 EMB samples following VP1/2 specific nested PCR. DNA (first lane) and cDNA (second lane) of each EMB were analysed. Amplicon length 173 bps EMBs 1, 6, 8, 9 and 10 were tested positive for viral DNA and negative for viral RNA. EMBs 3 and 5 were positive for both, viral RNA and DNA. EMBs 2, 4 and 7 were virus negative without any viral DNA nor RNA being detectable. M 100 bps marker, NC negative control, PC positive control.

    Journal: Scientific Reports

    Article Title: Detection of parvovirus mRNAs as markers for viral activity in endomyocardial biopsy-based diagnosis of patients with unexplained heart failure

    doi: 10.1038/s41598-020-78597-4

    Figure Lengend Snippet: ( A ) Schematic description of the B19V genome organization and PCR design. Gene locus and primer and probe localization for NS1 and VP1/2 are indicated by numbers representing the nucleotide position. ITR inverted terminal repeat, NS1 non structural protein 1, VP1/2 capsid proteins, P6 P6 promotor, (pA)p polyadenylation site proximal, (pA)d polyadenylation site distal. ( B ) Representative agarose gel electrophoresis gel blot image of a B19V-VP1/2 DNA and RNA-positive EMB using VP1/2 specific nested-PCR. Amplicon length 173 bps. 1 = DNA-Marker 100 bps; 2 = positive control; 3 = negative control; 4 = PCR after DNA extraction and DNAse treatment; 5 = PCR after RNA extraction, RNAse treatment and RT-PCR; 6 = PCR after DNA extraction; 7 = PCR after RNA extraction and DNAse treatment and RT-PCR. Complete gel blot image of figure ( B ) was shown in Supplementary Fig. S1 . ( C ) Representative agarose gel electrophoresis gel blot image of 10 EMB samples following VP1/2 specific nested PCR. DNA (first lane) and cDNA (second lane) of each EMB were analysed. Amplicon length 173 bps EMBs 1, 6, 8, 9 and 10 were tested positive for viral DNA and negative for viral RNA. EMBs 3 and 5 were positive for both, viral RNA and DNA. EMBs 2, 4 and 7 were virus negative without any viral DNA nor RNA being detectable. M 100 bps marker, NC negative control, PC positive control.

    Article Snippet: When both transcription intermediates (5156 ± 1076 GE/µg) or only VP1/2-RNA (5577 ± 1786 GE/µg) were detectable, the load of viral genomes was significantly increased compared to EMBs of patients that expressed solely NS1 RNA (2863 ± 454.2 GE/µg) (p = 0.0237; p = 0.0354) (Fig. C) Overall difference of viral load between these three groups was statistically significant (ANOVA p = 0.0427).

    Techniques: Polymerase Chain Reaction, Agarose Gel Electrophoresis, Western Blot, Nested PCR, Amplification, Marker, Positive Control, Negative Control, DNA Extraction, RNA Extraction, Reverse Transcription Polymerase Chain Reaction

    ( A ) B19V genome detection and detection of viral transcription activity in EMBs of patients with unexplained heart failure (N = 576). ( B ) The group composition of EMBs with detectable active viral transcription (VP1/2-RNA-, NS1-RNA and VP1/2 and NS1-RNA positive samples) was shown in detail. Numbers represent the amount of EMBs.

    Journal: Scientific Reports

    Article Title: Detection of parvovirus mRNAs as markers for viral activity in endomyocardial biopsy-based diagnosis of patients with unexplained heart failure

    doi: 10.1038/s41598-020-78597-4

    Figure Lengend Snippet: ( A ) B19V genome detection and detection of viral transcription activity in EMBs of patients with unexplained heart failure (N = 576). ( B ) The group composition of EMBs with detectable active viral transcription (VP1/2-RNA-, NS1-RNA and VP1/2 and NS1-RNA positive samples) was shown in detail. Numbers represent the amount of EMBs.

    Article Snippet: When both transcription intermediates (5156 ± 1076 GE/µg) or only VP1/2-RNA (5577 ± 1786 GE/µg) were detectable, the load of viral genomes was significantly increased compared to EMBs of patients that expressed solely NS1 RNA (2863 ± 454.2 GE/µg) (p = 0.0237; p = 0.0354) (Fig. C) Overall difference of viral load between these three groups was statistically significant (ANOVA p = 0.0427).

    Techniques: Activity Assay