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GE Healthcare glutathione sepharose 6b
LlmF interacts with VeA in the yeast-two-hybrid, in vitro GST pull-down, and in vivo co-purification. (A) A directed yeast-two-hybrid approach measured protein-protein interactions and indicated LlmF interacts with VeA, but not the truncated VeA1. Yeast cells harboring the indicated bait and prey plasmids were grown in liquid shaking culture to a density of approximately 2×10 7 cells/ml and 10 µl was spotted on synthetic dropout media (SD) containing the appropriate supplements (uracil (U), tryptophan (T), leucine (L), and/or X-gal). A positive interaction results in the activation of the lacZ reporter, which turns the media blue in the presence of X-gal. (B) Recombinant GST, GST-LlmF, GST-LaeA, and GST-VelB were incubated with recombinant His 6 -VeA-S-tag and subsequently purified by glutathione <t>sepharose</t> 6B to look for co-purification of VeA with any of the GST labeled proteins. An immunoblot using anti-S-tag antibody detected the presence of the His 6 -VeA-S-tag protein and Ponceau stain of the membrane served as an indication of the amount of GST fusion proteins in each lane. GST tagged LlmF, VelB, and LaeA were capable of pulling down His 6 -VeA-S-tag, while GST alone did not. (C) Crude protein extracts were prepared from one-liter liquid shaking culture of each strain and subjected to the TAP protein purification protocol. The resulting eluate was electrophoresed on a 10% Bis-Tris SDS-PAGE gel and transferred to a nitrocellulose membrane where an anti-calmodulin antibody confirmed TAP-LlmF and an anti-S-tag antibody was used to detect VeA-S-tag and VeA1-S-tag. Strains used are: WT = RJMP103.5, OE-TAP- llmF veA -S-tag = RJMP249.1, and OE-TAP- llmF veA1 -S-tag = RJMP250.2.
Glutathione Sepharose 6b, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 95/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Secondary Metabolism and Development Is Mediated by LlmF Control of VeA Subcellular Localization in Aspergillus nidulans"

Article Title: Secondary Metabolism and Development Is Mediated by LlmF Control of VeA Subcellular Localization in Aspergillus nidulans

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1003193

LlmF interacts with VeA in the yeast-two-hybrid, in vitro GST pull-down, and in vivo co-purification. (A) A directed yeast-two-hybrid approach measured protein-protein interactions and indicated LlmF interacts with VeA, but not the truncated VeA1. Yeast cells harboring the indicated bait and prey plasmids were grown in liquid shaking culture to a density of approximately 2×10 7 cells/ml and 10 µl was spotted on synthetic dropout media (SD) containing the appropriate supplements (uracil (U), tryptophan (T), leucine (L), and/or X-gal). A positive interaction results in the activation of the lacZ reporter, which turns the media blue in the presence of X-gal. (B) Recombinant GST, GST-LlmF, GST-LaeA, and GST-VelB were incubated with recombinant His 6 -VeA-S-tag and subsequently purified by glutathione sepharose 6B to look for co-purification of VeA with any of the GST labeled proteins. An immunoblot using anti-S-tag antibody detected the presence of the His 6 -VeA-S-tag protein and Ponceau stain of the membrane served as an indication of the amount of GST fusion proteins in each lane. GST tagged LlmF, VelB, and LaeA were capable of pulling down His 6 -VeA-S-tag, while GST alone did not. (C) Crude protein extracts were prepared from one-liter liquid shaking culture of each strain and subjected to the TAP protein purification protocol. The resulting eluate was electrophoresed on a 10% Bis-Tris SDS-PAGE gel and transferred to a nitrocellulose membrane where an anti-calmodulin antibody confirmed TAP-LlmF and an anti-S-tag antibody was used to detect VeA-S-tag and VeA1-S-tag. Strains used are: WT = RJMP103.5, OE-TAP- llmF veA -S-tag = RJMP249.1, and OE-TAP- llmF veA1 -S-tag = RJMP250.2.
Figure Legend Snippet: LlmF interacts with VeA in the yeast-two-hybrid, in vitro GST pull-down, and in vivo co-purification. (A) A directed yeast-two-hybrid approach measured protein-protein interactions and indicated LlmF interacts with VeA, but not the truncated VeA1. Yeast cells harboring the indicated bait and prey plasmids were grown in liquid shaking culture to a density of approximately 2×10 7 cells/ml and 10 µl was spotted on synthetic dropout media (SD) containing the appropriate supplements (uracil (U), tryptophan (T), leucine (L), and/or X-gal). A positive interaction results in the activation of the lacZ reporter, which turns the media blue in the presence of X-gal. (B) Recombinant GST, GST-LlmF, GST-LaeA, and GST-VelB were incubated with recombinant His 6 -VeA-S-tag and subsequently purified by glutathione sepharose 6B to look for co-purification of VeA with any of the GST labeled proteins. An immunoblot using anti-S-tag antibody detected the presence of the His 6 -VeA-S-tag protein and Ponceau stain of the membrane served as an indication of the amount of GST fusion proteins in each lane. GST tagged LlmF, VelB, and LaeA were capable of pulling down His 6 -VeA-S-tag, while GST alone did not. (C) Crude protein extracts were prepared from one-liter liquid shaking culture of each strain and subjected to the TAP protein purification protocol. The resulting eluate was electrophoresed on a 10% Bis-Tris SDS-PAGE gel and transferred to a nitrocellulose membrane where an anti-calmodulin antibody confirmed TAP-LlmF and an anti-S-tag antibody was used to detect VeA-S-tag and VeA1-S-tag. Strains used are: WT = RJMP103.5, OE-TAP- llmF veA -S-tag = RJMP249.1, and OE-TAP- llmF veA1 -S-tag = RJMP250.2.

Techniques Used: In Vitro, In Vivo, Copurification, Activation Assay, Recombinant, Incubation, Purification, Labeling, Staining, Protein Purification, SDS Page

2) Product Images from "Secondary Metabolism and Development Is Mediated by LlmF Control of VeA Subcellular Localization in Aspergillus nidulans"

Article Title: Secondary Metabolism and Development Is Mediated by LlmF Control of VeA Subcellular Localization in Aspergillus nidulans

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1003193

LlmF interacts with VeA in the yeast-two-hybrid, in vitro GST pull-down, and in vivo co-purification. (A) A directed yeast-two-hybrid approach measured protein-protein interactions and indicated LlmF interacts with VeA, but not the truncated VeA1. Yeast cells harboring the indicated bait and prey plasmids were grown in liquid shaking culture to a density of approximately 2×10 7 cells/ml and 10 µl was spotted on synthetic dropout media (SD) containing the appropriate supplements (uracil (U), tryptophan (T), leucine (L), and/or X-gal). A positive interaction results in the activation of the lacZ reporter, which turns the media blue in the presence of X-gal. (B) Recombinant GST, GST-LlmF, GST-LaeA, and GST-VelB were incubated with recombinant His 6 -VeA-S-tag and subsequently purified by glutathione sepharose 6B to look for co-purification of VeA with any of the GST labeled proteins. An immunoblot using anti-S-tag antibody detected the presence of the His 6 -VeA-S-tag protein and Ponceau stain of the membrane served as an indication of the amount of GST fusion proteins in each lane. GST tagged LlmF, VelB, and LaeA were capable of pulling down His 6 -VeA-S-tag, while GST alone did not. (C) Crude protein extracts were prepared from one-liter liquid shaking culture of each strain and subjected to the TAP protein purification protocol. The resulting eluate was electrophoresed on a 10% Bis-Tris SDS-PAGE gel and transferred to a nitrocellulose membrane where an anti-calmodulin antibody confirmed TAP-LlmF and an anti-S-tag antibody was used to detect VeA-S-tag and VeA1-S-tag. Strains used are: WT = RJMP103.5, OE-TAP- llmF veA -S-tag = RJMP249.1, and OE-TAP- llmF veA1 -S-tag = RJMP250.2.
Figure Legend Snippet: LlmF interacts with VeA in the yeast-two-hybrid, in vitro GST pull-down, and in vivo co-purification. (A) A directed yeast-two-hybrid approach measured protein-protein interactions and indicated LlmF interacts with VeA, but not the truncated VeA1. Yeast cells harboring the indicated bait and prey plasmids were grown in liquid shaking culture to a density of approximately 2×10 7 cells/ml and 10 µl was spotted on synthetic dropout media (SD) containing the appropriate supplements (uracil (U), tryptophan (T), leucine (L), and/or X-gal). A positive interaction results in the activation of the lacZ reporter, which turns the media blue in the presence of X-gal. (B) Recombinant GST, GST-LlmF, GST-LaeA, and GST-VelB were incubated with recombinant His 6 -VeA-S-tag and subsequently purified by glutathione sepharose 6B to look for co-purification of VeA with any of the GST labeled proteins. An immunoblot using anti-S-tag antibody detected the presence of the His 6 -VeA-S-tag protein and Ponceau stain of the membrane served as an indication of the amount of GST fusion proteins in each lane. GST tagged LlmF, VelB, and LaeA were capable of pulling down His 6 -VeA-S-tag, while GST alone did not. (C) Crude protein extracts were prepared from one-liter liquid shaking culture of each strain and subjected to the TAP protein purification protocol. The resulting eluate was electrophoresed on a 10% Bis-Tris SDS-PAGE gel and transferred to a nitrocellulose membrane where an anti-calmodulin antibody confirmed TAP-LlmF and an anti-S-tag antibody was used to detect VeA-S-tag and VeA1-S-tag. Strains used are: WT = RJMP103.5, OE-TAP- llmF veA -S-tag = RJMP249.1, and OE-TAP- llmF veA1 -S-tag = RJMP250.2.

Techniques Used: In Vitro, In Vivo, Copurification, Activation Assay, Recombinant, Incubation, Purification, Labeling, Staining, Protein Purification, SDS Page

Related Articles

Affinity Chromatography:

Article Title: A simple approach for preparation of affinity matrices: Simultaneous purification and reversible immobilization of a streptavidin mutein to agarose matrix
Article Snippet: .. L-ABD(KC) and SAVSBPM18-L-ABD(KC) in the dialyzed samples were purified by affinity chromatography using Sepharose 6B-CL (GE Healthcare Life Sciences). .. Bound proteins were eluted with TBS containing 20 mM lactose (Sigma).

Chromatography:

Article Title: Fractionation and identification of the allergic proteins in Aspergillus species
Article Snippet: .. The chromatography column was filled with DEAE-Sepharose 6 B resin (Amersham, Sweden). ..

Construct:

Article Title: Secondary Metabolism and Development Is Mediated by LlmF Control of VeA Subcellular Localization in Aspergillus nidulans
Article Snippet: .. A C terminal GST tag from pJMP89 was inserted into the XhoI site of pJMP126 to construct pJMP134 (His6 -VeA-GST-S-tag), which was subsequently tandem purified with Ni-NTA resin followed by glutathione sepharose 6B (GE healthcare). ..

Purification:

Article Title: A simple approach for preparation of affinity matrices: Simultaneous purification and reversible immobilization of a streptavidin mutein to agarose matrix
Article Snippet: .. L-ABD(KC) and SAVSBPM18-L-ABD(KC) in the dialyzed samples were purified by affinity chromatography using Sepharose 6B-CL (GE Healthcare Life Sciences). .. Bound proteins were eluted with TBS containing 20 mM lactose (Sigma).

Article Title: Secondary Metabolism and Development Is Mediated by LlmF Control of VeA Subcellular Localization in Aspergillus nidulans
Article Snippet: .. A C terminal GST tag from pJMP89 was inserted into the XhoI site of pJMP126 to construct pJMP134 (His6 -VeA-GST-S-tag), which was subsequently tandem purified with Ni-NTA resin followed by glutathione sepharose 6B (GE healthcare). ..

Incubation:

Article Title: Detection of Serum Thermolabile ?-2 Macroglycoprotein (Hakata Antigen) by Enzyme-Linked Immunosorbent Assay Using Polysaccharide Produced by Aerococcus viridans
Article Snippet: .. Sepharose 6B bead-conjugated PSA was prepared by the incubation of 1 mg of PSA and 0.3 g (dry weight) of epoxy-activated Sepharose 6B (Amersham Pharmacia Biotech) according to the manufacturer's instructions. .. After being washed extensively with PBS, the beads were mixed with 30 μl of SDS-PAGE sample buffer (250 mM Tris-HCl buffer [pH 6.7], 4% SDS, 0.05% bromophenol blue, and 50% glycerol) in the presence of 10 mM β-mercaptoethanol, incubated for 2 min at 100°C, and applied to SDS-PAGE (10% acrylamide).

other:

Article Title: Pu-Erh Tea Extract Induces the Degradation of FET Family Proteins Involved in the Pathogenesis of Amyotrophic Lateral Sclerosis
Article Snippet: Preparation of Tea Extract Sepharose 6B Beads Epoxy-activated Sepharose 6B beads were purchased from GE Healthcare Bio-Sciences Corporation (Little Chalfont, UK).

Microscopy:

Article Title: A simple approach for preparation of affinity matrices: Simultaneous purification and reversible immobilization of a streptavidin mutein to agarose matrix
Article Snippet: .. Microscopy of the distribution of Alexa Fluor 594 labelled L-ABD(KC) and its fusion on Sepharose 6B-CL Three 150-μl Sepharose 6B-CL columns were packed and loaded with Alexa Fluor 594, Alexa Fluor 594 labelled L-ABD(KC) and Alexa Fluor 594 labelled SAVSBPM18-L-ABD(KC), respectively. .. Beads taken at each time point were mounted onto slides and examined under Axio Image Z.1 microscope equipped with AxioVision (version 4.9.1.0) software (Zeiss).

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    GE Healthcare glutathione sepharose 6b
    LlmF interacts with VeA in the yeast-two-hybrid, in vitro GST pull-down, and in vivo co-purification. (A) A directed yeast-two-hybrid approach measured protein-protein interactions and indicated LlmF interacts with VeA, but not the truncated VeA1. Yeast cells harboring the indicated bait and prey plasmids were grown in liquid shaking culture to a density of approximately 2×10 7 cells/ml and 10 µl was spotted on synthetic dropout media (SD) containing the appropriate supplements (uracil (U), tryptophan (T), leucine (L), and/or X-gal). A positive interaction results in the activation of the lacZ reporter, which turns the media blue in the presence of X-gal. (B) Recombinant GST, GST-LlmF, GST-LaeA, and GST-VelB were incubated with recombinant His 6 -VeA-S-tag and subsequently purified by glutathione <t>sepharose</t> 6B to look for co-purification of VeA with any of the GST labeled proteins. An immunoblot using anti-S-tag antibody detected the presence of the His 6 -VeA-S-tag protein and Ponceau stain of the membrane served as an indication of the amount of GST fusion proteins in each lane. GST tagged LlmF, VelB, and LaeA were capable of pulling down His 6 -VeA-S-tag, while GST alone did not. (C) Crude protein extracts were prepared from one-liter liquid shaking culture of each strain and subjected to the TAP protein purification protocol. The resulting eluate was electrophoresed on a 10% Bis-Tris SDS-PAGE gel and transferred to a nitrocellulose membrane where an anti-calmodulin antibody confirmed TAP-LlmF and an anti-S-tag antibody was used to detect VeA-S-tag and VeA1-S-tag. Strains used are: WT = RJMP103.5, OE-TAP- llmF veA -S-tag = RJMP249.1, and OE-TAP- llmF veA1 -S-tag = RJMP250.2.
    Glutathione Sepharose 6b, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 95/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/glutathione sepharose 6b/product/GE Healthcare
    Average 95 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    glutathione sepharose 6b - by Bioz Stars, 2020-09
    95/100 stars
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    LlmF interacts with VeA in the yeast-two-hybrid, in vitro GST pull-down, and in vivo co-purification. (A) A directed yeast-two-hybrid approach measured protein-protein interactions and indicated LlmF interacts with VeA, but not the truncated VeA1. Yeast cells harboring the indicated bait and prey plasmids were grown in liquid shaking culture to a density of approximately 2×10 7 cells/ml and 10 µl was spotted on synthetic dropout media (SD) containing the appropriate supplements (uracil (U), tryptophan (T), leucine (L), and/or X-gal). A positive interaction results in the activation of the lacZ reporter, which turns the media blue in the presence of X-gal. (B) Recombinant GST, GST-LlmF, GST-LaeA, and GST-VelB were incubated with recombinant His 6 -VeA-S-tag and subsequently purified by glutathione sepharose 6B to look for co-purification of VeA with any of the GST labeled proteins. An immunoblot using anti-S-tag antibody detected the presence of the His 6 -VeA-S-tag protein and Ponceau stain of the membrane served as an indication of the amount of GST fusion proteins in each lane. GST tagged LlmF, VelB, and LaeA were capable of pulling down His 6 -VeA-S-tag, while GST alone did not. (C) Crude protein extracts were prepared from one-liter liquid shaking culture of each strain and subjected to the TAP protein purification protocol. The resulting eluate was electrophoresed on a 10% Bis-Tris SDS-PAGE gel and transferred to a nitrocellulose membrane where an anti-calmodulin antibody confirmed TAP-LlmF and an anti-S-tag antibody was used to detect VeA-S-tag and VeA1-S-tag. Strains used are: WT = RJMP103.5, OE-TAP- llmF veA -S-tag = RJMP249.1, and OE-TAP- llmF veA1 -S-tag = RJMP250.2.

    Journal: PLoS Genetics

    Article Title: Secondary Metabolism and Development Is Mediated by LlmF Control of VeA Subcellular Localization in Aspergillus nidulans

    doi: 10.1371/journal.pgen.1003193

    Figure Lengend Snippet: LlmF interacts with VeA in the yeast-two-hybrid, in vitro GST pull-down, and in vivo co-purification. (A) A directed yeast-two-hybrid approach measured protein-protein interactions and indicated LlmF interacts with VeA, but not the truncated VeA1. Yeast cells harboring the indicated bait and prey plasmids were grown in liquid shaking culture to a density of approximately 2×10 7 cells/ml and 10 µl was spotted on synthetic dropout media (SD) containing the appropriate supplements (uracil (U), tryptophan (T), leucine (L), and/or X-gal). A positive interaction results in the activation of the lacZ reporter, which turns the media blue in the presence of X-gal. (B) Recombinant GST, GST-LlmF, GST-LaeA, and GST-VelB were incubated with recombinant His 6 -VeA-S-tag and subsequently purified by glutathione sepharose 6B to look for co-purification of VeA with any of the GST labeled proteins. An immunoblot using anti-S-tag antibody detected the presence of the His 6 -VeA-S-tag protein and Ponceau stain of the membrane served as an indication of the amount of GST fusion proteins in each lane. GST tagged LlmF, VelB, and LaeA were capable of pulling down His 6 -VeA-S-tag, while GST alone did not. (C) Crude protein extracts were prepared from one-liter liquid shaking culture of each strain and subjected to the TAP protein purification protocol. The resulting eluate was electrophoresed on a 10% Bis-Tris SDS-PAGE gel and transferred to a nitrocellulose membrane where an anti-calmodulin antibody confirmed TAP-LlmF and an anti-S-tag antibody was used to detect VeA-S-tag and VeA1-S-tag. Strains used are: WT = RJMP103.5, OE-TAP- llmF veA -S-tag = RJMP249.1, and OE-TAP- llmF veA1 -S-tag = RJMP250.2.

    Article Snippet: A C terminal GST tag from pJMP89 was inserted into the XhoI site of pJMP126 to construct pJMP134 (His6 -VeA-GST-S-tag), which was subsequently tandem purified with Ni-NTA resin followed by glutathione sepharose 6B (GE healthcare).

    Techniques: In Vitro, In Vivo, Copurification, Activation Assay, Recombinant, Incubation, Purification, Labeling, Staining, Protein Purification, SDS Page