Structured Review

GE Healthcare glutathione sepharose 4ff
Siah-1 interacts with and ubiquitinates TRF2. ( a ) Siah-1 interacts with TRF2 in vitro . His 6 -TRF2 was immobilized on Ni-NTA magnetic agarose beads (lanes 3–5) and incubated with GST-Siah-1 (lane 4), GST alone (lane 5) or no additional protein (lane 3). In lane 2, GST-Siah-1 was incubated with Ni-NTA magnetic agarose beads without His 6 -tagged TRF2. After extensive washing, the beads were boiled in SDS sample buffer and the eluted proteins were analyzed by immunoblot using anti-GST and anti-TRF2 antibodies. In lanes 1, 7 and 8, His 6 -TRF2, GST-Siah-1 and GST alone were run directly as input controls. A molecular weight marker was in lane 6 (MW). ( b ) Siah-1 ubiquitinates TRF2 in vitro in a RING finger-dependent manner. Rabbit reticulocyte lysates (RRL), ubiquitin, an E3 ubiquitin ligase (wild-type Siah-1, Siah-1-H59W or Siah-1-ΔRING) and a substrate (GST-TRF2 or GST) were added to the in vitro ubiquitination reaction as indicated. After reaction, <t>glutathione-Sepharose</t> <t>4FF-purified</t> substrates were analyzed by immunoblot with anti-GST antibody. The position of non-ubiquitinated GST-TRF2 is indicated. Poly-ubiquitinated GST-TRF2 showed a smear signal (bracket) with the disappearance of non-ubiquitinated GST-TRF2. The experiment was repeated twice with reproducible results. ( c ) Siah-1 is essential to TRF2 ubiquitination in vivo . Myc-tagged TRF2, HA-tagged ubiquitin (Ub) and full-length p53 were transiently expressed in 293T cells, as indicated, which were pre-treated with control siRNA (−) or Siah-1 siRNA (#1 and #2). After treatment with MG132, protein lysates were prepared, immunoprecipitated with anti-Myc antibody or control IgG, and then analyzed by immunoblot using anti-Myc antibody (upper) and anti-HA antibody (lower). The knockdown of Siah-1 protein expression by Siah-1 siRNA was confirmed by immunoblot using total protein lysates before immunoprecipitation.β-actin was a loading control. White brackets indicate smear signals showing poly-ubiquitination. The strong signals at the bottom of the upper image correspond to IgG heavy chains. In the lower image, asterisks indicate non-specific bands. The closed arrowhead corresponded to the frontline of the electrophoresis, which likely contained non-specific signals and TRF2-associated ubiquitinated proteins of smaller size. The open arrowhead indicates a ubiquitinated protein of currently unknown origin. The experiment was repeated twice with reproducible results.
Glutathione Sepharose 4ff, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "Positive feedback between p53 and TRF2 in telomere damage signaling and cellular senescence"

Article Title: Positive feedback between p53 and TRF2 in telomere damage signaling and cellular senescence

Journal: Nature cell biology

doi: 10.1038/ncb2123

Siah-1 interacts with and ubiquitinates TRF2. ( a ) Siah-1 interacts with TRF2 in vitro . His 6 -TRF2 was immobilized on Ni-NTA magnetic agarose beads (lanes 3–5) and incubated with GST-Siah-1 (lane 4), GST alone (lane 5) or no additional protein (lane 3). In lane 2, GST-Siah-1 was incubated with Ni-NTA magnetic agarose beads without His 6 -tagged TRF2. After extensive washing, the beads were boiled in SDS sample buffer and the eluted proteins were analyzed by immunoblot using anti-GST and anti-TRF2 antibodies. In lanes 1, 7 and 8, His 6 -TRF2, GST-Siah-1 and GST alone were run directly as input controls. A molecular weight marker was in lane 6 (MW). ( b ) Siah-1 ubiquitinates TRF2 in vitro in a RING finger-dependent manner. Rabbit reticulocyte lysates (RRL), ubiquitin, an E3 ubiquitin ligase (wild-type Siah-1, Siah-1-H59W or Siah-1-ΔRING) and a substrate (GST-TRF2 or GST) were added to the in vitro ubiquitination reaction as indicated. After reaction, glutathione-Sepharose 4FF-purified substrates were analyzed by immunoblot with anti-GST antibody. The position of non-ubiquitinated GST-TRF2 is indicated. Poly-ubiquitinated GST-TRF2 showed a smear signal (bracket) with the disappearance of non-ubiquitinated GST-TRF2. The experiment was repeated twice with reproducible results. ( c ) Siah-1 is essential to TRF2 ubiquitination in vivo . Myc-tagged TRF2, HA-tagged ubiquitin (Ub) and full-length p53 were transiently expressed in 293T cells, as indicated, which were pre-treated with control siRNA (−) or Siah-1 siRNA (#1 and #2). After treatment with MG132, protein lysates were prepared, immunoprecipitated with anti-Myc antibody or control IgG, and then analyzed by immunoblot using anti-Myc antibody (upper) and anti-HA antibody (lower). The knockdown of Siah-1 protein expression by Siah-1 siRNA was confirmed by immunoblot using total protein lysates before immunoprecipitation.β-actin was a loading control. White brackets indicate smear signals showing poly-ubiquitination. The strong signals at the bottom of the upper image correspond to IgG heavy chains. In the lower image, asterisks indicate non-specific bands. The closed arrowhead corresponded to the frontline of the electrophoresis, which likely contained non-specific signals and TRF2-associated ubiquitinated proteins of smaller size. The open arrowhead indicates a ubiquitinated protein of currently unknown origin. The experiment was repeated twice with reproducible results.
Figure Legend Snippet: Siah-1 interacts with and ubiquitinates TRF2. ( a ) Siah-1 interacts with TRF2 in vitro . His 6 -TRF2 was immobilized on Ni-NTA magnetic agarose beads (lanes 3–5) and incubated with GST-Siah-1 (lane 4), GST alone (lane 5) or no additional protein (lane 3). In lane 2, GST-Siah-1 was incubated with Ni-NTA magnetic agarose beads without His 6 -tagged TRF2. After extensive washing, the beads were boiled in SDS sample buffer and the eluted proteins were analyzed by immunoblot using anti-GST and anti-TRF2 antibodies. In lanes 1, 7 and 8, His 6 -TRF2, GST-Siah-1 and GST alone were run directly as input controls. A molecular weight marker was in lane 6 (MW). ( b ) Siah-1 ubiquitinates TRF2 in vitro in a RING finger-dependent manner. Rabbit reticulocyte lysates (RRL), ubiquitin, an E3 ubiquitin ligase (wild-type Siah-1, Siah-1-H59W or Siah-1-ΔRING) and a substrate (GST-TRF2 or GST) were added to the in vitro ubiquitination reaction as indicated. After reaction, glutathione-Sepharose 4FF-purified substrates were analyzed by immunoblot with anti-GST antibody. The position of non-ubiquitinated GST-TRF2 is indicated. Poly-ubiquitinated GST-TRF2 showed a smear signal (bracket) with the disappearance of non-ubiquitinated GST-TRF2. The experiment was repeated twice with reproducible results. ( c ) Siah-1 is essential to TRF2 ubiquitination in vivo . Myc-tagged TRF2, HA-tagged ubiquitin (Ub) and full-length p53 were transiently expressed in 293T cells, as indicated, which were pre-treated with control siRNA (−) or Siah-1 siRNA (#1 and #2). After treatment with MG132, protein lysates were prepared, immunoprecipitated with anti-Myc antibody or control IgG, and then analyzed by immunoblot using anti-Myc antibody (upper) and anti-HA antibody (lower). The knockdown of Siah-1 protein expression by Siah-1 siRNA was confirmed by immunoblot using total protein lysates before immunoprecipitation.β-actin was a loading control. White brackets indicate smear signals showing poly-ubiquitination. The strong signals at the bottom of the upper image correspond to IgG heavy chains. In the lower image, asterisks indicate non-specific bands. The closed arrowhead corresponded to the frontline of the electrophoresis, which likely contained non-specific signals and TRF2-associated ubiquitinated proteins of smaller size. The open arrowhead indicates a ubiquitinated protein of currently unknown origin. The experiment was repeated twice with reproducible results.

Techniques Used: In Vitro, Incubation, Molecular Weight, Marker, Purification, In Vivo, Immunoprecipitation, Expressing, Electrophoresis

2) Product Images from "Positive feedback between p53 and TRF2 in telomere damage signaling and cellular senescence"

Article Title: Positive feedback between p53 and TRF2 in telomere damage signaling and cellular senescence

Journal: Nature cell biology

doi: 10.1038/ncb2123

Siah-1 interacts with and ubiquitinates TRF2. ( a ) Siah-1 interacts with TRF2 in vitro . His 6 -TRF2 was immobilized on Ni-NTA magnetic agarose beads (lanes 3–5) and incubated with GST-Siah-1 (lane 4), GST alone (lane 5) or no additional protein (lane 3). In lane 2, GST-Siah-1 was incubated with Ni-NTA magnetic agarose beads without His 6 -tagged TRF2. After extensive washing, the beads were boiled in SDS sample buffer and the eluted proteins were analyzed by immunoblot using anti-GST and anti-TRF2 antibodies. In lanes 1, 7 and 8, His 6 -TRF2, GST-Siah-1 and GST alone were run directly as input controls. A molecular weight marker was in lane 6 (MW). ( b ) Siah-1 ubiquitinates TRF2 in vitro in a RING finger-dependent manner. Rabbit reticulocyte lysates (RRL), ubiquitin, an E3 ubiquitin ligase (wild-type Siah-1, Siah-1-H59W or Siah-1-ΔRING) and a substrate (GST-TRF2 or GST) were added to the in vitro ubiquitination reaction as indicated. After reaction, glutathione-Sepharose 4FF-purified substrates were analyzed by immunoblot with anti-GST antibody. The position of non-ubiquitinated GST-TRF2 is indicated. Poly-ubiquitinated GST-TRF2 showed a smear signal (bracket) with the disappearance of non-ubiquitinated GST-TRF2. The experiment was repeated twice with reproducible results. ( c ) Siah-1 is essential to TRF2 ubiquitination in vivo . Myc-tagged TRF2, HA-tagged ubiquitin (Ub) and full-length p53 were transiently expressed in 293T cells, as indicated, which were pre-treated with control siRNA (−) or Siah-1 siRNA (#1 and #2). After treatment with MG132, protein lysates were prepared, immunoprecipitated with anti-Myc antibody or control IgG, and then analyzed by immunoblot using anti-Myc antibody (upper) and anti-HA antibody (lower). The knockdown of Siah-1 protein expression by Siah-1 siRNA was confirmed by immunoblot using total protein lysates before immunoprecipitation.β-actin was a loading control. White brackets indicate smear signals showing poly-ubiquitination. The strong signals at the bottom of the upper image correspond to IgG heavy chains. In the lower image, asterisks indicate non-specific bands. The closed arrowhead corresponded to the frontline of the electrophoresis, which likely contained non-specific signals and TRF2-associated ubiquitinated proteins of smaller size. The open arrowhead indicates a ubiquitinated protein of currently unknown origin. The experiment was repeated twice with reproducible results.
Figure Legend Snippet: Siah-1 interacts with and ubiquitinates TRF2. ( a ) Siah-1 interacts with TRF2 in vitro . His 6 -TRF2 was immobilized on Ni-NTA magnetic agarose beads (lanes 3–5) and incubated with GST-Siah-1 (lane 4), GST alone (lane 5) or no additional protein (lane 3). In lane 2, GST-Siah-1 was incubated with Ni-NTA magnetic agarose beads without His 6 -tagged TRF2. After extensive washing, the beads were boiled in SDS sample buffer and the eluted proteins were analyzed by immunoblot using anti-GST and anti-TRF2 antibodies. In lanes 1, 7 and 8, His 6 -TRF2, GST-Siah-1 and GST alone were run directly as input controls. A molecular weight marker was in lane 6 (MW). ( b ) Siah-1 ubiquitinates TRF2 in vitro in a RING finger-dependent manner. Rabbit reticulocyte lysates (RRL), ubiquitin, an E3 ubiquitin ligase (wild-type Siah-1, Siah-1-H59W or Siah-1-ΔRING) and a substrate (GST-TRF2 or GST) were added to the in vitro ubiquitination reaction as indicated. After reaction, glutathione-Sepharose 4FF-purified substrates were analyzed by immunoblot with anti-GST antibody. The position of non-ubiquitinated GST-TRF2 is indicated. Poly-ubiquitinated GST-TRF2 showed a smear signal (bracket) with the disappearance of non-ubiquitinated GST-TRF2. The experiment was repeated twice with reproducible results. ( c ) Siah-1 is essential to TRF2 ubiquitination in vivo . Myc-tagged TRF2, HA-tagged ubiquitin (Ub) and full-length p53 were transiently expressed in 293T cells, as indicated, which were pre-treated with control siRNA (−) or Siah-1 siRNA (#1 and #2). After treatment with MG132, protein lysates were prepared, immunoprecipitated with anti-Myc antibody or control IgG, and then analyzed by immunoblot using anti-Myc antibody (upper) and anti-HA antibody (lower). The knockdown of Siah-1 protein expression by Siah-1 siRNA was confirmed by immunoblot using total protein lysates before immunoprecipitation.β-actin was a loading control. White brackets indicate smear signals showing poly-ubiquitination. The strong signals at the bottom of the upper image correspond to IgG heavy chains. In the lower image, asterisks indicate non-specific bands. The closed arrowhead corresponded to the frontline of the electrophoresis, which likely contained non-specific signals and TRF2-associated ubiquitinated proteins of smaller size. The open arrowhead indicates a ubiquitinated protein of currently unknown origin. The experiment was repeated twice with reproducible results.

Techniques Used: In Vitro, Incubation, Molecular Weight, Marker, Purification, In Vivo, Immunoprecipitation, Expressing, Electrophoresis

3) Product Images from "Positive feedback between p53 and TRF2 in telomere damage signaling and cellular senescence"

Article Title: Positive feedback between p53 and TRF2 in telomere damage signaling and cellular senescence

Journal: Nature cell biology

doi: 10.1038/ncb2123

Siah-1 interacts with and ubiquitinates TRF2. ( a ) Siah-1 interacts with TRF2 in vitro . His 6 -TRF2 was immobilized on Ni-NTA magnetic agarose beads (lanes 3–5) and incubated with GST-Siah-1 (lane 4), GST alone (lane 5) or no additional protein (lane 3). In lane 2, GST-Siah-1 was incubated with Ni-NTA magnetic agarose beads without His 6 -tagged TRF2. After extensive washing, the beads were boiled in SDS sample buffer and the eluted proteins were analyzed by immunoblot using anti-GST and anti-TRF2 antibodies. In lanes 1, 7 and 8, His 6 -TRF2, GST-Siah-1 and GST alone were run directly as input controls. A molecular weight marker was in lane 6 (MW). ( b ) Siah-1 ubiquitinates TRF2 in vitro in a RING finger-dependent manner. Rabbit reticulocyte lysates (RRL), ubiquitin, an E3 ubiquitin ligase (wild-type Siah-1, Siah-1-H59W or Siah-1-ΔRING) and a substrate (GST-TRF2 or GST) were added to the in vitro ubiquitination reaction as indicated. After reaction, glutathione-Sepharose 4FF-purified substrates were analyzed by immunoblot with anti-GST antibody. The position of non-ubiquitinated GST-TRF2 is indicated. Poly-ubiquitinated GST-TRF2 showed a smear signal (bracket) with the disappearance of non-ubiquitinated GST-TRF2. The experiment was repeated twice with reproducible results. ( c ) Siah-1 is essential to TRF2 ubiquitination in vivo . Myc-tagged TRF2, HA-tagged ubiquitin (Ub) and full-length p53 were transiently expressed in 293T cells, as indicated, which were pre-treated with control siRNA (−) or Siah-1 siRNA (#1 and #2). After treatment with MG132, protein lysates were prepared, immunoprecipitated with anti-Myc antibody or control IgG, and then analyzed by immunoblot using anti-Myc antibody (upper) and anti-HA antibody (lower). The knockdown of Siah-1 protein expression by Siah-1 siRNA was confirmed by immunoblot using total protein lysates before immunoprecipitation.β-actin was a loading control. White brackets indicate smear signals showing poly-ubiquitination. The strong signals at the bottom of the upper image correspond to IgG heavy chains. In the lower image, asterisks indicate non-specific bands. The closed arrowhead corresponded to the frontline of the electrophoresis, which likely contained non-specific signals and TRF2-associated ubiquitinated proteins of smaller size. The open arrowhead indicates a ubiquitinated protein of currently unknown origin. The experiment was repeated twice with reproducible results.
Figure Legend Snippet: Siah-1 interacts with and ubiquitinates TRF2. ( a ) Siah-1 interacts with TRF2 in vitro . His 6 -TRF2 was immobilized on Ni-NTA magnetic agarose beads (lanes 3–5) and incubated with GST-Siah-1 (lane 4), GST alone (lane 5) or no additional protein (lane 3). In lane 2, GST-Siah-1 was incubated with Ni-NTA magnetic agarose beads without His 6 -tagged TRF2. After extensive washing, the beads were boiled in SDS sample buffer and the eluted proteins were analyzed by immunoblot using anti-GST and anti-TRF2 antibodies. In lanes 1, 7 and 8, His 6 -TRF2, GST-Siah-1 and GST alone were run directly as input controls. A molecular weight marker was in lane 6 (MW). ( b ) Siah-1 ubiquitinates TRF2 in vitro in a RING finger-dependent manner. Rabbit reticulocyte lysates (RRL), ubiquitin, an E3 ubiquitin ligase (wild-type Siah-1, Siah-1-H59W or Siah-1-ΔRING) and a substrate (GST-TRF2 or GST) were added to the in vitro ubiquitination reaction as indicated. After reaction, glutathione-Sepharose 4FF-purified substrates were analyzed by immunoblot with anti-GST antibody. The position of non-ubiquitinated GST-TRF2 is indicated. Poly-ubiquitinated GST-TRF2 showed a smear signal (bracket) with the disappearance of non-ubiquitinated GST-TRF2. The experiment was repeated twice with reproducible results. ( c ) Siah-1 is essential to TRF2 ubiquitination in vivo . Myc-tagged TRF2, HA-tagged ubiquitin (Ub) and full-length p53 were transiently expressed in 293T cells, as indicated, which were pre-treated with control siRNA (−) or Siah-1 siRNA (#1 and #2). After treatment with MG132, protein lysates were prepared, immunoprecipitated with anti-Myc antibody or control IgG, and then analyzed by immunoblot using anti-Myc antibody (upper) and anti-HA antibody (lower). The knockdown of Siah-1 protein expression by Siah-1 siRNA was confirmed by immunoblot using total protein lysates before immunoprecipitation.β-actin was a loading control. White brackets indicate smear signals showing poly-ubiquitination. The strong signals at the bottom of the upper image correspond to IgG heavy chains. In the lower image, asterisks indicate non-specific bands. The closed arrowhead corresponded to the frontline of the electrophoresis, which likely contained non-specific signals and TRF2-associated ubiquitinated proteins of smaller size. The open arrowhead indicates a ubiquitinated protein of currently unknown origin. The experiment was repeated twice with reproducible results.

Techniques Used: In Vitro, Incubation, Molecular Weight, Marker, Purification, In Vivo, Immunoprecipitation, Expressing, Electrophoresis

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    GE Healthcare glutathione sepharose 4ff
    Siah-1 interacts with and ubiquitinates TRF2. ( a ) Siah-1 interacts with TRF2 in vitro . His 6 -TRF2 was immobilized on Ni-NTA magnetic agarose beads (lanes 3–5) and incubated with GST-Siah-1 (lane 4), GST alone (lane 5) or no additional protein (lane 3). In lane 2, GST-Siah-1 was incubated with Ni-NTA magnetic agarose beads without His 6 -tagged TRF2. After extensive washing, the beads were boiled in SDS sample buffer and the eluted proteins were analyzed by immunoblot using anti-GST and anti-TRF2 antibodies. In lanes 1, 7 and 8, His 6 -TRF2, GST-Siah-1 and GST alone were run directly as input controls. A molecular weight marker was in lane 6 (MW). ( b ) Siah-1 ubiquitinates TRF2 in vitro in a RING finger-dependent manner. Rabbit reticulocyte lysates (RRL), ubiquitin, an E3 ubiquitin ligase (wild-type Siah-1, Siah-1-H59W or Siah-1-ΔRING) and a substrate (GST-TRF2 or GST) were added to the in vitro ubiquitination reaction as indicated. After reaction, <t>glutathione-Sepharose</t> <t>4FF-purified</t> substrates were analyzed by immunoblot with anti-GST antibody. The position of non-ubiquitinated GST-TRF2 is indicated. Poly-ubiquitinated GST-TRF2 showed a smear signal (bracket) with the disappearance of non-ubiquitinated GST-TRF2. The experiment was repeated twice with reproducible results. ( c ) Siah-1 is essential to TRF2 ubiquitination in vivo . Myc-tagged TRF2, HA-tagged ubiquitin (Ub) and full-length p53 were transiently expressed in 293T cells, as indicated, which were pre-treated with control siRNA (−) or Siah-1 siRNA (#1 and #2). After treatment with MG132, protein lysates were prepared, immunoprecipitated with anti-Myc antibody or control IgG, and then analyzed by immunoblot using anti-Myc antibody (upper) and anti-HA antibody (lower). The knockdown of Siah-1 protein expression by Siah-1 siRNA was confirmed by immunoblot using total protein lysates before immunoprecipitation.β-actin was a loading control. White brackets indicate smear signals showing poly-ubiquitination. The strong signals at the bottom of the upper image correspond to IgG heavy chains. In the lower image, asterisks indicate non-specific bands. The closed arrowhead corresponded to the frontline of the electrophoresis, which likely contained non-specific signals and TRF2-associated ubiquitinated proteins of smaller size. The open arrowhead indicates a ubiquitinated protein of currently unknown origin. The experiment was repeated twice with reproducible results.
    Glutathione Sepharose 4ff, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/glutathione sepharose 4ff/product/GE Healthcare
    Average 99 stars, based on 12 article reviews
    Price from $9.99 to $1999.99
    glutathione sepharose 4ff - by Bioz Stars, 2020-09
    99/100 stars
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    Siah-1 interacts with and ubiquitinates TRF2. ( a ) Siah-1 interacts with TRF2 in vitro . His 6 -TRF2 was immobilized on Ni-NTA magnetic agarose beads (lanes 3–5) and incubated with GST-Siah-1 (lane 4), GST alone (lane 5) or no additional protein (lane 3). In lane 2, GST-Siah-1 was incubated with Ni-NTA magnetic agarose beads without His 6 -tagged TRF2. After extensive washing, the beads were boiled in SDS sample buffer and the eluted proteins were analyzed by immunoblot using anti-GST and anti-TRF2 antibodies. In lanes 1, 7 and 8, His 6 -TRF2, GST-Siah-1 and GST alone were run directly as input controls. A molecular weight marker was in lane 6 (MW). ( b ) Siah-1 ubiquitinates TRF2 in vitro in a RING finger-dependent manner. Rabbit reticulocyte lysates (RRL), ubiquitin, an E3 ubiquitin ligase (wild-type Siah-1, Siah-1-H59W or Siah-1-ΔRING) and a substrate (GST-TRF2 or GST) were added to the in vitro ubiquitination reaction as indicated. After reaction, glutathione-Sepharose 4FF-purified substrates were analyzed by immunoblot with anti-GST antibody. The position of non-ubiquitinated GST-TRF2 is indicated. Poly-ubiquitinated GST-TRF2 showed a smear signal (bracket) with the disappearance of non-ubiquitinated GST-TRF2. The experiment was repeated twice with reproducible results. ( c ) Siah-1 is essential to TRF2 ubiquitination in vivo . Myc-tagged TRF2, HA-tagged ubiquitin (Ub) and full-length p53 were transiently expressed in 293T cells, as indicated, which were pre-treated with control siRNA (−) or Siah-1 siRNA (#1 and #2). After treatment with MG132, protein lysates were prepared, immunoprecipitated with anti-Myc antibody or control IgG, and then analyzed by immunoblot using anti-Myc antibody (upper) and anti-HA antibody (lower). The knockdown of Siah-1 protein expression by Siah-1 siRNA was confirmed by immunoblot using total protein lysates before immunoprecipitation.β-actin was a loading control. White brackets indicate smear signals showing poly-ubiquitination. The strong signals at the bottom of the upper image correspond to IgG heavy chains. In the lower image, asterisks indicate non-specific bands. The closed arrowhead corresponded to the frontline of the electrophoresis, which likely contained non-specific signals and TRF2-associated ubiquitinated proteins of smaller size. The open arrowhead indicates a ubiquitinated protein of currently unknown origin. The experiment was repeated twice with reproducible results.

    Journal: Nature cell biology

    Article Title: Positive feedback between p53 and TRF2 in telomere damage signaling and cellular senescence

    doi: 10.1038/ncb2123

    Figure Lengend Snippet: Siah-1 interacts with and ubiquitinates TRF2. ( a ) Siah-1 interacts with TRF2 in vitro . His 6 -TRF2 was immobilized on Ni-NTA magnetic agarose beads (lanes 3–5) and incubated with GST-Siah-1 (lane 4), GST alone (lane 5) or no additional protein (lane 3). In lane 2, GST-Siah-1 was incubated with Ni-NTA magnetic agarose beads without His 6 -tagged TRF2. After extensive washing, the beads were boiled in SDS sample buffer and the eluted proteins were analyzed by immunoblot using anti-GST and anti-TRF2 antibodies. In lanes 1, 7 and 8, His 6 -TRF2, GST-Siah-1 and GST alone were run directly as input controls. A molecular weight marker was in lane 6 (MW). ( b ) Siah-1 ubiquitinates TRF2 in vitro in a RING finger-dependent manner. Rabbit reticulocyte lysates (RRL), ubiquitin, an E3 ubiquitin ligase (wild-type Siah-1, Siah-1-H59W or Siah-1-ΔRING) and a substrate (GST-TRF2 or GST) were added to the in vitro ubiquitination reaction as indicated. After reaction, glutathione-Sepharose 4FF-purified substrates were analyzed by immunoblot with anti-GST antibody. The position of non-ubiquitinated GST-TRF2 is indicated. Poly-ubiquitinated GST-TRF2 showed a smear signal (bracket) with the disappearance of non-ubiquitinated GST-TRF2. The experiment was repeated twice with reproducible results. ( c ) Siah-1 is essential to TRF2 ubiquitination in vivo . Myc-tagged TRF2, HA-tagged ubiquitin (Ub) and full-length p53 were transiently expressed in 293T cells, as indicated, which were pre-treated with control siRNA (−) or Siah-1 siRNA (#1 and #2). After treatment with MG132, protein lysates were prepared, immunoprecipitated with anti-Myc antibody or control IgG, and then analyzed by immunoblot using anti-Myc antibody (upper) and anti-HA antibody (lower). The knockdown of Siah-1 protein expression by Siah-1 siRNA was confirmed by immunoblot using total protein lysates before immunoprecipitation.β-actin was a loading control. White brackets indicate smear signals showing poly-ubiquitination. The strong signals at the bottom of the upper image correspond to IgG heavy chains. In the lower image, asterisks indicate non-specific bands. The closed arrowhead corresponded to the frontline of the electrophoresis, which likely contained non-specific signals and TRF2-associated ubiquitinated proteins of smaller size. The open arrowhead indicates a ubiquitinated protein of currently unknown origin. The experiment was repeated twice with reproducible results.

    Article Snippet: In vitro protein synthesis of His6 -tagged TRF2, GST and GST-Siah-1 fusion protein was performed using Expressway cell-free E. coli expression system (Invitrogen), followed by the purification of His6 -tagged TRF2 using Ni-NTA magnetic agarose beads (Qiagen, Valencia, CA) and of GST and GST-Siah-1 using glutathione-Sepharose 4FF (GE Healthcare, Piscataway, NJ).

    Techniques: In Vitro, Incubation, Molecular Weight, Marker, Purification, In Vivo, Immunoprecipitation, Expressing, Electrophoresis