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GE Healthcare glutathione sepharose 4b column
Interaction between the NF90ctv RG- domain and Rev by affinity chromatography. (A) The GST/RG- recombinant protein was expressed in E. coli , the cell extracts coupled to a <t>glutathione-Sepharose</t> <t>4B</t> column was used in pull-down assays with HeLa cell extracts previously transfected with pRSV/Rev (lane 4) or the control lysate (lane 3). A protein band corresponding to Rev (arrowhead, lane 4) was detected when extracts from HeLa cells that expressed Rev were added to the GST/RG- protein bound column; such a band was absent from control HeLa cell extract (lane 3). Lane 1, GST/RG- purified from E. coli induced by IPTG, and lane 2, purified from non induced E. coli cells. (B) Similar assays performed with purified Rev protein from E. coli in place of HeLa cells extracts. Arrow in lane 5 indicates the position of Rev. This band was not observed in absence of Rev (lane 6).
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1) Product Images from "Nuclear Factor 90, a cellular dsRNA binding protein inhibits the HIV Rev-export function"

Article Title: Nuclear Factor 90, a cellular dsRNA binding protein inhibits the HIV Rev-export function

Journal: Retrovirology

doi: 10.1186/1742-4690-3-83

Interaction between the NF90ctv RG- domain and Rev by affinity chromatography. (A) The GST/RG- recombinant protein was expressed in E. coli , the cell extracts coupled to a glutathione-Sepharose 4B column was used in pull-down assays with HeLa cell extracts previously transfected with pRSV/Rev (lane 4) or the control lysate (lane 3). A protein band corresponding to Rev (arrowhead, lane 4) was detected when extracts from HeLa cells that expressed Rev were added to the GST/RG- protein bound column; such a band was absent from control HeLa cell extract (lane 3). Lane 1, GST/RG- purified from E. coli induced by IPTG, and lane 2, purified from non induced E. coli cells. (B) Similar assays performed with purified Rev protein from E. coli in place of HeLa cells extracts. Arrow in lane 5 indicates the position of Rev. This band was not observed in absence of Rev (lane 6).
Figure Legend Snippet: Interaction between the NF90ctv RG- domain and Rev by affinity chromatography. (A) The GST/RG- recombinant protein was expressed in E. coli , the cell extracts coupled to a glutathione-Sepharose 4B column was used in pull-down assays with HeLa cell extracts previously transfected with pRSV/Rev (lane 4) or the control lysate (lane 3). A protein band corresponding to Rev (arrowhead, lane 4) was detected when extracts from HeLa cells that expressed Rev were added to the GST/RG- protein bound column; such a band was absent from control HeLa cell extract (lane 3). Lane 1, GST/RG- purified from E. coli induced by IPTG, and lane 2, purified from non induced E. coli cells. (B) Similar assays performed with purified Rev protein from E. coli in place of HeLa cells extracts. Arrow in lane 5 indicates the position of Rev. This band was not observed in absence of Rev (lane 6).

Techniques Used: Affinity Chromatography, Recombinant, Transfection, Purification

2) Product Images from "PfMSA180 is a novel Plasmodium falciparum vaccine antigen that interacts with human erythrocyte integrin associated protein (CD47)"

Article Title: PfMSA180 is a novel Plasmodium falciparum vaccine antigen that interacts with human erythrocyte integrin associated protein (CD47)

Journal: Scientific Reports

doi: 10.1038/s41598-019-42366-9

PfMSA180 (PF3D7_1014100) exists as a 170 kDa protein in parasites. ( A ) Schematic representation of full-length PfMSA180 and recombinant protein fragments (Tr 1–5) PfMSA180 consists of 1455 aa with a calculated molecular mass (MW) of 173.3 kDa. The protein has a predicted signal peptide (SP; 1 to 22 aa; shown in green). Recombinant PfMSA180 truncates were expressed as N-terminal GST-tagged proteins by the wheat germ cell-free system (WGCFS). Tr1, residues E 22 -S 263 (expected MW 29 kDa); Tr2, A 264 -D 501 (expected MW 27.9 kDa); Tr3, I 508 -P 723 (expected MW 52.8 kDa, including the GST tag); Tr4, A 805 -P 1093 (expected MW 34 0.4 kDa); and Tr5, L 1193 -P 1455 (expected MW 58.5 kDa including the GST tag). ( B ) Recombinant PfMSA180 truncates are shown stained with Coomassie brilliant blue (CBB) following purification on a glutathione-Sepharose 4B column and resolution by 12.5% SDS-PAGE under reducing conditions. Arrowheads indicate molecular masses predicted from amino acid sequences of the corresponding recombinant PfMSA180 truncates (original data is included in the Supplementary Data File 1 ). ( C ) Reactivity of rabbit anti-PfMSA180 antibodies to native PfMSA180. Schizont-rich parasite pellets were solubilized with NP40 and PfMSA180 immunoprecipitated with the indicated mouse antibodies. Mouse anti-HisGST antibody was used as a negative control. Immunoprecipitated full length PfMSA180 was detected using rabbit antibody to each of the truncates; indicated using an arrowhead. The additional bands observed in the membrane fraction at 120, 80, and 45 kDa, were likely products of protein SUB1-mediated proteolysis of full-length protein. Antibodies to truncate 3 did not immunoprecipitate protein (original data is included in the Supplementary Data File 1 ).
Figure Legend Snippet: PfMSA180 (PF3D7_1014100) exists as a 170 kDa protein in parasites. ( A ) Schematic representation of full-length PfMSA180 and recombinant protein fragments (Tr 1–5) PfMSA180 consists of 1455 aa with a calculated molecular mass (MW) of 173.3 kDa. The protein has a predicted signal peptide (SP; 1 to 22 aa; shown in green). Recombinant PfMSA180 truncates were expressed as N-terminal GST-tagged proteins by the wheat germ cell-free system (WGCFS). Tr1, residues E 22 -S 263 (expected MW 29 kDa); Tr2, A 264 -D 501 (expected MW 27.9 kDa); Tr3, I 508 -P 723 (expected MW 52.8 kDa, including the GST tag); Tr4, A 805 -P 1093 (expected MW 34 0.4 kDa); and Tr5, L 1193 -P 1455 (expected MW 58.5 kDa including the GST tag). ( B ) Recombinant PfMSA180 truncates are shown stained with Coomassie brilliant blue (CBB) following purification on a glutathione-Sepharose 4B column and resolution by 12.5% SDS-PAGE under reducing conditions. Arrowheads indicate molecular masses predicted from amino acid sequences of the corresponding recombinant PfMSA180 truncates (original data is included in the Supplementary Data File 1 ). ( C ) Reactivity of rabbit anti-PfMSA180 antibodies to native PfMSA180. Schizont-rich parasite pellets were solubilized with NP40 and PfMSA180 immunoprecipitated with the indicated mouse antibodies. Mouse anti-HisGST antibody was used as a negative control. Immunoprecipitated full length PfMSA180 was detected using rabbit antibody to each of the truncates; indicated using an arrowhead. The additional bands observed in the membrane fraction at 120, 80, and 45 kDa, were likely products of protein SUB1-mediated proteolysis of full-length protein. Antibodies to truncate 3 did not immunoprecipitate protein (original data is included in the Supplementary Data File 1 ).

Techniques Used: Recombinant, Staining, Purification, SDS Page, Immunoprecipitation, Negative Control

3) Product Images from "A novel function of N-linked glycoproteins, alpha-2-HS-glycoprotein and hemopexin: Implications for small molecule compound-mediated neuroprotection"

Article Title: A novel function of N-linked glycoproteins, alpha-2-HS-glycoprotein and hemopexin: Implications for small molecule compound-mediated neuroprotection

Journal: PLoS ONE

doi: 10.1371/journal.pone.0186227

SDS-PAGE profiles of human WN1316-activating factor candidates synthesized in the cell-free system. The reaction mixture of GST protein synthesis (crude) and the GST protein after glutathione-Sepharose 4B column purification (puri.) were resolved on a 5–20% gradient SDS–PAGE gel, and gel was stained with Coomassie blue. Black arrowheads indicate synthesized GST fusion WN1316-activating factors. White arrowhead indicates GST used as a negative control.
Figure Legend Snippet: SDS-PAGE profiles of human WN1316-activating factor candidates synthesized in the cell-free system. The reaction mixture of GST protein synthesis (crude) and the GST protein after glutathione-Sepharose 4B column purification (puri.) were resolved on a 5–20% gradient SDS–PAGE gel, and gel was stained with Coomassie blue. Black arrowheads indicate synthesized GST fusion WN1316-activating factors. White arrowhead indicates GST used as a negative control.

Techniques Used: SDS Page, Synthesized, Purification, Staining, Negative Control

4) Product Images from "PfMSA180 is a novel Plasmodium falciparum vaccine antigen that interacts with human erythrocyte integrin associated protein (CD47)"

Article Title: PfMSA180 is a novel Plasmodium falciparum vaccine antigen that interacts with human erythrocyte integrin associated protein (CD47)

Journal: Scientific Reports

doi: 10.1038/s41598-019-42366-9

PfMSA180 (PF3D7_1014100) exists as a 170 kDa protein in parasites. ( A ) Schematic representation of full-length PfMSA180 and recombinant protein fragments (Tr 1–5) PfMSA180 consists of 1455 aa with a calculated molecular mass (MW) of 173.3 kDa. The protein has a predicted signal peptide (SP; 1 to 22 aa; shown in green). Recombinant PfMSA180 truncates were expressed as N-terminal GST-tagged proteins by the wheat germ cell-free system (WGCFS). Tr1, residues E 22 -S 263 (expected MW 29 kDa); Tr2, A 264 -D 501 (expected MW 27.9 kDa); Tr3, I 508 -P 723 (expected MW 52.8 kDa, including the GST tag); Tr4, A 805 -P 1093 (expected MW 34 0.4 kDa); and Tr5, L 1193 -P 1455 (expected MW 58.5 kDa including the GST tag). ( B ) Recombinant PfMSA180 truncates are shown stained with Coomassie brilliant blue (CBB) following purification on a glutathione-Sepharose 4B column and resolution by 12.5% SDS-PAGE under reducing conditions. Arrowheads indicate molecular masses predicted from amino acid sequences of the corresponding recombinant PfMSA180 truncates (original data is included in the Supplementary Data File 1 ). ( C ) Reactivity of rabbit anti-PfMSA180 antibodies to native PfMSA180. Schizont-rich parasite pellets were solubilized with NP40 and PfMSA180 immunoprecipitated with the indicated mouse antibodies. Mouse anti-HisGST antibody was used as a negative control. Immunoprecipitated full length PfMSA180 was detected using rabbit antibody to each of the truncates; indicated using an arrowhead. The additional bands observed in the membrane fraction at 120, 80, and 45 kDa, were likely products of protein SUB1-mediated proteolysis of full-length protein. Antibodies to truncate 3 did not immunoprecipitate protein (original data is included in the Supplementary Data File 1 ).
Figure Legend Snippet: PfMSA180 (PF3D7_1014100) exists as a 170 kDa protein in parasites. ( A ) Schematic representation of full-length PfMSA180 and recombinant protein fragments (Tr 1–5) PfMSA180 consists of 1455 aa with a calculated molecular mass (MW) of 173.3 kDa. The protein has a predicted signal peptide (SP; 1 to 22 aa; shown in green). Recombinant PfMSA180 truncates were expressed as N-terminal GST-tagged proteins by the wheat germ cell-free system (WGCFS). Tr1, residues E 22 -S 263 (expected MW 29 kDa); Tr2, A 264 -D 501 (expected MW 27.9 kDa); Tr3, I 508 -P 723 (expected MW 52.8 kDa, including the GST tag); Tr4, A 805 -P 1093 (expected MW 34 0.4 kDa); and Tr5, L 1193 -P 1455 (expected MW 58.5 kDa including the GST tag). ( B ) Recombinant PfMSA180 truncates are shown stained with Coomassie brilliant blue (CBB) following purification on a glutathione-Sepharose 4B column and resolution by 12.5% SDS-PAGE under reducing conditions. Arrowheads indicate molecular masses predicted from amino acid sequences of the corresponding recombinant PfMSA180 truncates (original data is included in the Supplementary Data File 1 ). ( C ) Reactivity of rabbit anti-PfMSA180 antibodies to native PfMSA180. Schizont-rich parasite pellets were solubilized with NP40 and PfMSA180 immunoprecipitated with the indicated mouse antibodies. Mouse anti-HisGST antibody was used as a negative control. Immunoprecipitated full length PfMSA180 was detected using rabbit antibody to each of the truncates; indicated using an arrowhead. The additional bands observed in the membrane fraction at 120, 80, and 45 kDa, were likely products of protein SUB1-mediated proteolysis of full-length protein. Antibodies to truncate 3 did not immunoprecipitate protein (original data is included in the Supplementary Data File 1 ).

Techniques Used: Recombinant, Staining, Purification, SDS Page, Immunoprecipitation, Negative Control

5) Product Images from "CiAPEX2 and CiP0, candidates of AP endonucleases in Ciona intestinalis, have 3′-5′ exonuclease activity and contribute to protection against oxidative stress"

Article Title: CiAPEX2 and CiP0, candidates of AP endonucleases in Ciona intestinalis, have 3′-5′ exonuclease activity and contribute to protection against oxidative stress

Journal: Genes and Environment

doi: 10.1186/s41021-017-0087-7

Identification of APEX2 and P0 homologues in C. intestinalis and purification results. a and b The amino acid sequences of C. intestinalis APEX2 and P0 are aligned with H. sapiens and S. cerevisiae homologues for APEX2 and H. sapiens and D. melanogaster homologues for P0. Amino acid residues are highlighted in black (identical) or grey (similar). a Amino acid sequence alignment among APEX2 homologues. Conserved endonuclease/exonuclease/phosphatase domain are enclosed by a black box. b Amino acid sequence alignment among P0 homologues. c - e Purification of GST-CiAPEX2 ( c ), GST-CiP0 ( d ), tag-free CiP0 ( d ) and His-CiAPEX2 ( e ). Each protein was purified from bacteria lysate using column chromatography. Purified proteins were electrophoresed on a 10% SDS-PAGE gel, and stained with Coomassie brilliant blue. The arrows indicate each of the purified proteins. Especially in ( d ), upper arrow indicates GST-CiP0 and lower arrow indicates tag-free CiP0. c and ( d GST-CiAPEX2 ( c ), GST-CiP0 ( d ) and tag-free CiP0 ( d ) purification. The fractions are as follows: SUP, supernatant; GST/FT, flow-through fraction from Glutathione-Sepharose 4B column; GST/Wash, washed fraction by buffer A; GST/Elu, fraction eluted by 40 mM glutathione; tag-free CiP0, the product of GST-CiP0 thrombin cleavage; Cleaved GST, the byproduct of GST-CiP0 thrombin cleavage. e His-CiAPEX2 purification. The fractions are as follows: SUP, supernatant; His/FT, flow-through fraction from Chelating Sepharose Fast Flow; His/ Wash, washed fraction by buffer D containing 10 mM imidazole; His/Elu, fraction eluted by 0.5 M imidazole; Q/ FT, flow-through fraction from HiTrap-Q; Q/0.235-0.32 M, fractions collected in 0.235-0.32 M NaCl
Figure Legend Snippet: Identification of APEX2 and P0 homologues in C. intestinalis and purification results. a and b The amino acid sequences of C. intestinalis APEX2 and P0 are aligned with H. sapiens and S. cerevisiae homologues for APEX2 and H. sapiens and D. melanogaster homologues for P0. Amino acid residues are highlighted in black (identical) or grey (similar). a Amino acid sequence alignment among APEX2 homologues. Conserved endonuclease/exonuclease/phosphatase domain are enclosed by a black box. b Amino acid sequence alignment among P0 homologues. c - e Purification of GST-CiAPEX2 ( c ), GST-CiP0 ( d ), tag-free CiP0 ( d ) and His-CiAPEX2 ( e ). Each protein was purified from bacteria lysate using column chromatography. Purified proteins were electrophoresed on a 10% SDS-PAGE gel, and stained with Coomassie brilliant blue. The arrows indicate each of the purified proteins. Especially in ( d ), upper arrow indicates GST-CiP0 and lower arrow indicates tag-free CiP0. c and ( d GST-CiAPEX2 ( c ), GST-CiP0 ( d ) and tag-free CiP0 ( d ) purification. The fractions are as follows: SUP, supernatant; GST/FT, flow-through fraction from Glutathione-Sepharose 4B column; GST/Wash, washed fraction by buffer A; GST/Elu, fraction eluted by 40 mM glutathione; tag-free CiP0, the product of GST-CiP0 thrombin cleavage; Cleaved GST, the byproduct of GST-CiP0 thrombin cleavage. e His-CiAPEX2 purification. The fractions are as follows: SUP, supernatant; His/FT, flow-through fraction from Chelating Sepharose Fast Flow; His/ Wash, washed fraction by buffer D containing 10 mM imidazole; His/Elu, fraction eluted by 0.5 M imidazole; Q/ FT, flow-through fraction from HiTrap-Q; Q/0.235-0.32 M, fractions collected in 0.235-0.32 M NaCl

Techniques Used: Purification, Sequencing, Column Chromatography, SDS Page, Staining, Flow Cytometry

6) Product Images from "A novel function of N-linked glycoproteins, alpha-2-HS-glycoprotein and hemopexin: Implications for small molecule compound-mediated neuroprotection"

Article Title: A novel function of N-linked glycoproteins, alpha-2-HS-glycoprotein and hemopexin: Implications for small molecule compound-mediated neuroprotection

Journal: PLoS ONE

doi: 10.1371/journal.pone.0186227

SDS-PAGE profiles of human WN1316-activating factor candidates synthesized in the cell-free system. The reaction mixture of GST protein synthesis (crude) and the GST protein after glutathione-Sepharose 4B column purification (puri.) were resolved on a 5–20% gradient SDS–PAGE gel, and gel was stained with Coomassie blue. Black arrowheads indicate synthesized GST fusion WN1316-activating factors. White arrowhead indicates GST used as a negative control.
Figure Legend Snippet: SDS-PAGE profiles of human WN1316-activating factor candidates synthesized in the cell-free system. The reaction mixture of GST protein synthesis (crude) and the GST protein after glutathione-Sepharose 4B column purification (puri.) were resolved on a 5–20% gradient SDS–PAGE gel, and gel was stained with Coomassie blue. Black arrowheads indicate synthesized GST fusion WN1316-activating factors. White arrowhead indicates GST used as a negative control.

Techniques Used: SDS Page, Synthesized, Purification, Staining, Negative Control

7) Product Images from "Molecular Cloning and Characterization of Taurocyamine Kinase from Clonorchis sinensis: A Candidate Chemotherapeutic Target"

Article Title: Molecular Cloning and Characterization of Taurocyamine Kinase from Clonorchis sinensis: A Candidate Chemotherapeutic Target

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0002548

Purification of recombinant CsTKD1 by on-bead cleavage. Cs28GST-CsTKD1 was loaded to a glutathione sepharose 4B column and cleaved with thrombin. The cleaved-off CsTKD1 was eluted with PBS.
Figure Legend Snippet: Purification of recombinant CsTKD1 by on-bead cleavage. Cs28GST-CsTKD1 was loaded to a glutathione sepharose 4B column and cleaved with thrombin. The cleaved-off CsTKD1 was eluted with PBS.

Techniques Used: Purification, Recombinant

8) Product Images from "CiAPEX2 and CiP0, candidates of AP endonucleases in Ciona intestinalis, have 3′-5′ exonuclease activity and contribute to protection against oxidative stress"

Article Title: CiAPEX2 and CiP0, candidates of AP endonucleases in Ciona intestinalis, have 3′-5′ exonuclease activity and contribute to protection against oxidative stress

Journal: Genes and Environment

doi: 10.1186/s41021-017-0087-7

Identification of APEX2 and P0 homologues in C. intestinalis and purification results. a and b The amino acid sequences of C. intestinalis APEX2 and P0 are aligned with H. sapiens and S. cerevisiae homologues for APEX2 and H. sapiens and D. melanogaster homologues for P0. Amino acid residues are highlighted in black (identical) or grey (similar). a Amino acid sequence alignment among APEX2 homologues. Conserved endonuclease/exonuclease/phosphatase domain are enclosed by a black box. b Amino acid sequence alignment among P0 homologues. c - e Purification of GST-CiAPEX2 ( c ), GST-CiP0 ( d ), tag-free CiP0 ( d ) and His-CiAPEX2 ( e ). Each protein was purified from bacteria lysate using column chromatography. Purified proteins were electrophoresed on a 10% SDS-PAGE gel, and stained with Coomassie brilliant blue. The arrows indicate each of the purified proteins. Especially in ( d ), upper arrow indicates GST-CiP0 and lower arrow indicates tag-free CiP0. c and ( d GST-CiAPEX2 ( c ), GST-CiP0 ( d ) and tag-free CiP0 ( d ) purification. The fractions are as follows: SUP, supernatant; GST/FT, flow-through fraction from Glutathione-Sepharose 4B column; GST/Wash, washed fraction by buffer A; GST/Elu, fraction eluted by 40 mM glutathione; tag-free CiP0, the product of GST-CiP0 thrombin cleavage; Cleaved GST, the byproduct of GST-CiP0 thrombin cleavage. e His-CiAPEX2 purification. The fractions are as follows: SUP, supernatant; His/FT, flow-through fraction from Chelating Sepharose Fast Flow; His/ Wash, washed fraction by buffer D containing 10 mM imidazole; His/Elu, fraction eluted by 0.5 M imidazole; Q/ FT, flow-through fraction from HiTrap-Q; Q/0.235-0.32 M, fractions collected in 0.235-0.32 M NaCl
Figure Legend Snippet: Identification of APEX2 and P0 homologues in C. intestinalis and purification results. a and b The amino acid sequences of C. intestinalis APEX2 and P0 are aligned with H. sapiens and S. cerevisiae homologues for APEX2 and H. sapiens and D. melanogaster homologues for P0. Amino acid residues are highlighted in black (identical) or grey (similar). a Amino acid sequence alignment among APEX2 homologues. Conserved endonuclease/exonuclease/phosphatase domain are enclosed by a black box. b Amino acid sequence alignment among P0 homologues. c - e Purification of GST-CiAPEX2 ( c ), GST-CiP0 ( d ), tag-free CiP0 ( d ) and His-CiAPEX2 ( e ). Each protein was purified from bacteria lysate using column chromatography. Purified proteins were electrophoresed on a 10% SDS-PAGE gel, and stained with Coomassie brilliant blue. The arrows indicate each of the purified proteins. Especially in ( d ), upper arrow indicates GST-CiP0 and lower arrow indicates tag-free CiP0. c and ( d GST-CiAPEX2 ( c ), GST-CiP0 ( d ) and tag-free CiP0 ( d ) purification. The fractions are as follows: SUP, supernatant; GST/FT, flow-through fraction from Glutathione-Sepharose 4B column; GST/Wash, washed fraction by buffer A; GST/Elu, fraction eluted by 40 mM glutathione; tag-free CiP0, the product of GST-CiP0 thrombin cleavage; Cleaved GST, the byproduct of GST-CiP0 thrombin cleavage. e His-CiAPEX2 purification. The fractions are as follows: SUP, supernatant; His/FT, flow-through fraction from Chelating Sepharose Fast Flow; His/ Wash, washed fraction by buffer D containing 10 mM imidazole; His/Elu, fraction eluted by 0.5 M imidazole; Q/ FT, flow-through fraction from HiTrap-Q; Q/0.235-0.32 M, fractions collected in 0.235-0.32 M NaCl

Techniques Used: Purification, Sequencing, Column Chromatography, SDS Page, Staining, Flow Cytometry

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Centrifugation:

Article Title: The WD40 protein Morg1 facilitates Par6-aPKC binding to Crb3 for apical identity in epithelial cells
Article Snippet: .. In brief, recombinant proteins were expressed in the E. coli strain BL21, and cells were homogenized by sonication; after centrifugation, GST- or MBP-tagged proteins were purified using glutathione-Sepharose 4B (GE Healthcare) or amylose resin (New England Biolabs, Inc.), respectively. .. Recombinant Cdc42 (Q61L) and Cdc42 (T17N), both of which lack the C-terminal residues 189–191 and carry the C188S substitution for protein stabilization, were expressed as GST fusion protein in the E. coli strain BL21, and proteins were applied to glutathione-Sepharose 4B.

Affinity Chromatography:

Article Title: A Docking Site in MKK4 Mediates High Affinity Binding to JNK MAPKs and Competes with Similar Docking Sites in JNK Substrates *
Article Snippet: .. GST fusion proteins were expressed in bacteria, purified by affinity chromatography using glutathione-Sepharose (Amersham Biosciences), and quantified as described ( ). ..

Purification:

Article Title: The WD40 protein Morg1 facilitates Par6-aPKC binding to Crb3 for apical identity in epithelial cells
Article Snippet: .. In brief, recombinant proteins were expressed in the E. coli strain BL21, and cells were homogenized by sonication; after centrifugation, GST- or MBP-tagged proteins were purified using glutathione-Sepharose 4B (GE Healthcare) or amylose resin (New England Biolabs, Inc.), respectively. .. Recombinant Cdc42 (Q61L) and Cdc42 (T17N), both of which lack the C-terminal residues 189–191 and carry the C188S substitution for protein stabilization, were expressed as GST fusion protein in the E. coli strain BL21, and proteins were applied to glutathione-Sepharose 4B.

Article Title: The Kinesin-associated Protein UNC-76 Is Required for Axonal Transport in the Drosophila Nervous System
Article Snippet: .. For affinity purification of UNC-76 antisera, purified pGEX-H4 protein was cross-linked to glutathione-Sepharose 4B (Amersham Biosciences, Piscataway, NJ) according to established protocols ( ). .. Affinity purified UNC-76 antisera were used at a 1:100 dilution for immunostaining and a 1:1000 dilution for Western blots.

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Expressing:

Article Title: Histone phosphorylation by TRPM6’s cleaved kinase attenuates adjacent arginine methylation to regulate gene expression
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Sonication:

Article Title: The WD40 protein Morg1 facilitates Par6-aPKC binding to Crb3 for apical identity in epithelial cells
Article Snippet: .. In brief, recombinant proteins were expressed in the E. coli strain BL21, and cells were homogenized by sonication; after centrifugation, GST- or MBP-tagged proteins were purified using glutathione-Sepharose 4B (GE Healthcare) or amylose resin (New England Biolabs, Inc.), respectively. .. Recombinant Cdc42 (Q61L) and Cdc42 (T17N), both of which lack the C-terminal residues 189–191 and carry the C188S substitution for protein stabilization, were expressed as GST fusion protein in the E. coli strain BL21, and proteins were applied to glutathione-Sepharose 4B.

Affinity Purification:

Article Title: The Kinesin-associated Protein UNC-76 Is Required for Axonal Transport in the Drosophila Nervous System
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Recombinant:

Article Title: The WD40 protein Morg1 facilitates Par6-aPKC binding to Crb3 for apical identity in epithelial cells
Article Snippet: .. In brief, recombinant proteins were expressed in the E. coli strain BL21, and cells were homogenized by sonication; after centrifugation, GST- or MBP-tagged proteins were purified using glutathione-Sepharose 4B (GE Healthcare) or amylose resin (New England Biolabs, Inc.), respectively. .. Recombinant Cdc42 (Q61L) and Cdc42 (T17N), both of which lack the C-terminal residues 189–191 and carry the C188S substitution for protein stabilization, were expressed as GST fusion protein in the E. coli strain BL21, and proteins were applied to glutathione-Sepharose 4B.

Article Title: Nuclear Factor 90, a cellular dsRNA binding protein inhibits the HIV Rev-export function
Article Snippet: .. The recombinant protein was purified using a glutathione-Sepharose 4B column (MicroSpin™ GST Purification Module, Amersham) following the manufacturer's instructions. .. The recombinant protein was verified by SDS-PAGE and by immunoblotting with α-GST (Amersham) or α-GST/RG- polyclonal antibodies.

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    GE Healthcare glutathione sepharose high performance column
    Comparison of C-terminal affinities for Tom70 between Tom20 and the chaperones. A , His-Tom70 ΔN (WT) was co-precipitated either with unloaded <t>glutathione-Sepharose</t> ( lane 4 ) or with purified GST fusion proteins (GST, lane 5 ; or GST-Tom20 ΔN
    Glutathione Sepharose High Performance Column, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 96/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GE Healthcare glutathione sepharose affinity column
    Enhancement of αa HEL protein RNA duplex unwinding by the γb protein. Panel A : SDS-PAGE and immunoblot analysis of purified MBP-αa HEL protein. The recombinant MBP-αa HEL protein containing a histidine tag and the αa helicase domain fused to the C-terminus of the maltose binding protein was expressed in E . coli strain BL21 and purified over Ni−NTA <t>agarose</t> columns. SDS-PAGE staining with Coomassie brilliant blue (CBB) is shown on the left side and immunoblot analysis (IB) with α-HIS antibodies is shown on the right side. Panel B: SDS-PAGE analysis of GST-tagged γb derivatives purified from E . coli . Sizes (in kDa) of molecular weight markers (Mr) are shown on the left sides of the blots. Panel C : Schematic depiction of the dsRNA substrate used for RNA unwinding assays. Preparation of the radioactive labeled substrate is described in the Materials and Methods and in S2 Table . The long and short thick lines indicate the template RNA strands, and vertical dashed lines represent regions of base pairing. The lengths of the double-stranded and overhanging portions of the RNA strands are given in base pairs (bp) and nucleotides (nt), respectively. Panel D. RNA helicase activities of the purified MBP-αa HEL and GST-γb proteins in the presence or absence of ATP. The mobility of the radioactive labeled RNAs was tested using native acrylamide gel electrophoresis followed by phosphor imaging. Lane 1, migration of the partial dsRNA band. Lane 2, migration of single-stranded RNA produced by heating the dsRNA substrate at 95°C for 5 min. Lane 3, Negative control showing lack of helicase activity of purified MBP. Lanes 4–8, RNA unwinding reactions containing increasing amounts of purified MBP-αa HEL (1.8–6.2 pmol) in the presence of 5 mM ATP. Lanes 9–11, RNA unwinding reactions containing increasing amounts of purified GST-γb (1.8–6.2 pmol) in the presence of 5 mM ATP. Lanes 12–13 indicate the RNA helicase activity of MBP-αa HEL with ATP or without (w/o) addition of ATP to the reaction mixture. Note: Lanes 1–8 and 9–13 were separated on separate gels. Panel E. dsRNA unwinding of MBP-αa HEL in RNA helicase reactions containing wt GST-γb, GST-γb BM26 or GST. Helicase assays were carried out with 4.4 pmol of the MBP-αa HEL protein in the presence of increasing concentrations (0.5, 3.0, 6.0 pM) of GST (lanes 3–5), γb (lanes 6–8, 12–14) or γb BM26 (lanes 9–11) respectively. Positions of the partial dsRNA substrate and the released 55 nt 32 P ssRNA strand product are indicated on the left (lanes 1–2). Note: Lanes 1–8 and 9–14 were separated on separate gels. Panel F: Quantification of dsRNA unwinding efficiency shown in Fig 7E. The results are plotted as percentages of ssRNA release compared to the ssRNA in lane 2.
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    Image Search Results


    Comparison of C-terminal affinities for Tom70 between Tom20 and the chaperones. A , His-Tom70 ΔN (WT) was co-precipitated either with unloaded glutathione-Sepharose ( lane 4 ) or with purified GST fusion proteins (GST, lane 5 ; or GST-Tom20 ΔN

    Journal: The Journal of Biological Chemistry

    Article Title: Interaction between the Human Mitochondrial Import Receptors Tom20 and Tom70 in Vitro Suggests a Chaperone Displacement Mechanism *

    doi: 10.1074/jbc.M111.280446

    Figure Lengend Snippet: Comparison of C-terminal affinities for Tom70 between Tom20 and the chaperones. A , His-Tom70 ΔN (WT) was co-precipitated either with unloaded glutathione-Sepharose ( lane 4 ) or with purified GST fusion proteins (GST, lane 5 ; or GST-Tom20 ΔN

    Article Snippet: The GST-tagged proteins were bound to a glutathione-Sepharose high performance column (GE Healthcare) equilibrated in PBS and eluted with PBS and 20 m m glutathione.

    Techniques: Purification

    Two regions of Tom20 are responsible for the interaction with Tom70. A , His-Tom70 ΔN (WT or R192A) was co-precipitated either with unloaded glutathione-Sepharose ( lanes 5 and 6 ) or with purified GST fusion proteins (GST, lanes 7 and 8 ; or GST-Tom20

    Journal: The Journal of Biological Chemistry

    Article Title: Interaction between the Human Mitochondrial Import Receptors Tom20 and Tom70 in Vitro Suggests a Chaperone Displacement Mechanism *

    doi: 10.1074/jbc.M111.280446

    Figure Lengend Snippet: Two regions of Tom20 are responsible for the interaction with Tom70. A , His-Tom70 ΔN (WT or R192A) was co-precipitated either with unloaded glutathione-Sepharose ( lanes 5 and 6 ) or with purified GST fusion proteins (GST, lanes 7 and 8 ; or GST-Tom20

    Article Snippet: The GST-tagged proteins were bound to a glutathione-Sepharose high performance column (GE Healthcare) equilibrated in PBS and eluted with PBS and 20 m m glutathione.

    Techniques: Purification

    Enhancement of αa HEL protein RNA duplex unwinding by the γb protein. Panel A : SDS-PAGE and immunoblot analysis of purified MBP-αa HEL protein. The recombinant MBP-αa HEL protein containing a histidine tag and the αa helicase domain fused to the C-terminus of the maltose binding protein was expressed in E . coli strain BL21 and purified over Ni−NTA agarose columns. SDS-PAGE staining with Coomassie brilliant blue (CBB) is shown on the left side and immunoblot analysis (IB) with α-HIS antibodies is shown on the right side. Panel B: SDS-PAGE analysis of GST-tagged γb derivatives purified from E . coli . Sizes (in kDa) of molecular weight markers (Mr) are shown on the left sides of the blots. Panel C : Schematic depiction of the dsRNA substrate used for RNA unwinding assays. Preparation of the radioactive labeled substrate is described in the Materials and Methods and in S2 Table . The long and short thick lines indicate the template RNA strands, and vertical dashed lines represent regions of base pairing. The lengths of the double-stranded and overhanging portions of the RNA strands are given in base pairs (bp) and nucleotides (nt), respectively. Panel D. RNA helicase activities of the purified MBP-αa HEL and GST-γb proteins in the presence or absence of ATP. The mobility of the radioactive labeled RNAs was tested using native acrylamide gel electrophoresis followed by phosphor imaging. Lane 1, migration of the partial dsRNA band. Lane 2, migration of single-stranded RNA produced by heating the dsRNA substrate at 95°C for 5 min. Lane 3, Negative control showing lack of helicase activity of purified MBP. Lanes 4–8, RNA unwinding reactions containing increasing amounts of purified MBP-αa HEL (1.8–6.2 pmol) in the presence of 5 mM ATP. Lanes 9–11, RNA unwinding reactions containing increasing amounts of purified GST-γb (1.8–6.2 pmol) in the presence of 5 mM ATP. Lanes 12–13 indicate the RNA helicase activity of MBP-αa HEL with ATP or without (w/o) addition of ATP to the reaction mixture. Note: Lanes 1–8 and 9–13 were separated on separate gels. Panel E. dsRNA unwinding of MBP-αa HEL in RNA helicase reactions containing wt GST-γb, GST-γb BM26 or GST. Helicase assays were carried out with 4.4 pmol of the MBP-αa HEL protein in the presence of increasing concentrations (0.5, 3.0, 6.0 pM) of GST (lanes 3–5), γb (lanes 6–8, 12–14) or γb BM26 (lanes 9–11) respectively. Positions of the partial dsRNA substrate and the released 55 nt 32 P ssRNA strand product are indicated on the left (lanes 1–2). Note: Lanes 1–8 and 9–14 were separated on separate gels. Panel F: Quantification of dsRNA unwinding efficiency shown in Fig 7E. The results are plotted as percentages of ssRNA release compared to the ssRNA in lane 2.

    Journal: PLoS Pathogens

    Article Title: The Barley stripe mosaic virus γb protein promotes chloroplast-targeted replication by enhancing unwinding of RNA duplexes

    doi: 10.1371/journal.ppat.1006319

    Figure Lengend Snippet: Enhancement of αa HEL protein RNA duplex unwinding by the γb protein. Panel A : SDS-PAGE and immunoblot analysis of purified MBP-αa HEL protein. The recombinant MBP-αa HEL protein containing a histidine tag and the αa helicase domain fused to the C-terminus of the maltose binding protein was expressed in E . coli strain BL21 and purified over Ni−NTA agarose columns. SDS-PAGE staining with Coomassie brilliant blue (CBB) is shown on the left side and immunoblot analysis (IB) with α-HIS antibodies is shown on the right side. Panel B: SDS-PAGE analysis of GST-tagged γb derivatives purified from E . coli . Sizes (in kDa) of molecular weight markers (Mr) are shown on the left sides of the blots. Panel C : Schematic depiction of the dsRNA substrate used for RNA unwinding assays. Preparation of the radioactive labeled substrate is described in the Materials and Methods and in S2 Table . The long and short thick lines indicate the template RNA strands, and vertical dashed lines represent regions of base pairing. The lengths of the double-stranded and overhanging portions of the RNA strands are given in base pairs (bp) and nucleotides (nt), respectively. Panel D. RNA helicase activities of the purified MBP-αa HEL and GST-γb proteins in the presence or absence of ATP. The mobility of the radioactive labeled RNAs was tested using native acrylamide gel electrophoresis followed by phosphor imaging. Lane 1, migration of the partial dsRNA band. Lane 2, migration of single-stranded RNA produced by heating the dsRNA substrate at 95°C for 5 min. Lane 3, Negative control showing lack of helicase activity of purified MBP. Lanes 4–8, RNA unwinding reactions containing increasing amounts of purified MBP-αa HEL (1.8–6.2 pmol) in the presence of 5 mM ATP. Lanes 9–11, RNA unwinding reactions containing increasing amounts of purified GST-γb (1.8–6.2 pmol) in the presence of 5 mM ATP. Lanes 12–13 indicate the RNA helicase activity of MBP-αa HEL with ATP or without (w/o) addition of ATP to the reaction mixture. Note: Lanes 1–8 and 9–13 were separated on separate gels. Panel E. dsRNA unwinding of MBP-αa HEL in RNA helicase reactions containing wt GST-γb, GST-γb BM26 or GST. Helicase assays were carried out with 4.4 pmol of the MBP-αa HEL protein in the presence of increasing concentrations (0.5, 3.0, 6.0 pM) of GST (lanes 3–5), γb (lanes 6–8, 12–14) or γb BM26 (lanes 9–11) respectively. Positions of the partial dsRNA substrate and the released 55 nt 32 P ssRNA strand product are indicated on the left (lanes 1–2). Note: Lanes 1–8 and 9–14 were separated on separate gels. Panel F: Quantification of dsRNA unwinding efficiency shown in Fig 7E. The results are plotted as percentages of ssRNA release compared to the ssRNA in lane 2.

    Article Snippet: For recovery of GST-tagged proteins, supernatants recovered after centrifugation were passed over a glutathione-Sepharose affinity column (GE Healthcare), and the GST fusion proteins were eluted with T buffer containing 60 mM L-Glutathione and 2 mM DTT.

    Techniques: SDS Page, Purification, Recombinant, Binding Assay, Staining, Molecular Weight, Labeling, Acrylamide Gel Assay, Electrophoresis, Imaging, Migration, Produced, Negative Control, Activity Assay

    SPAK and OSR1 chimera and inhibitory action of SPAK fragments on NKCC2 function. A , different SPAK and OSR1 chimera were built and purified using a glutathione-Sepharose column. All chimeras have the PF2 domain, required for detection by the C-terminal

    Journal: The Journal of Biological Chemistry

    Article Title: Short Forms of Ste20-related Proline/Alanine-rich Kinase (SPAK) in the Kidney Are Created by Aspartyl Aminopeptidase (Dnpep)-mediated Proteolytic Cleavage

    doi: 10.1074/jbc.M114.604009

    Figure Lengend Snippet: SPAK and OSR1 chimera and inhibitory action of SPAK fragments on NKCC2 function. A , different SPAK and OSR1 chimera were built and purified using a glutathione-Sepharose column. All chimeras have the PF2 domain, required for detection by the C-terminal

    Article Snippet: Supernatant was loaded onto a glutathione-Sepharose column (GE Healthcare).

    Techniques: Purification

    Dnpep fusion protein cleaves SPAK fusion protein. A , reaction between GST-SPAK and GST-Dnpep. GST-Dnpep fusion protein ( FP ) was detected in the supernatant of lysed Dnpep-transformed bacteria and purified by a glutathione-Sepharose column. SPAK fusion

    Journal: The Journal of Biological Chemistry

    Article Title: Short Forms of Ste20-related Proline/Alanine-rich Kinase (SPAK) in the Kidney Are Created by Aspartyl Aminopeptidase (Dnpep)-mediated Proteolytic Cleavage

    doi: 10.1074/jbc.M114.604009

    Figure Lengend Snippet: Dnpep fusion protein cleaves SPAK fusion protein. A , reaction between GST-SPAK and GST-Dnpep. GST-Dnpep fusion protein ( FP ) was detected in the supernatant of lysed Dnpep-transformed bacteria and purified by a glutathione-Sepharose column. SPAK fusion

    Article Snippet: Supernatant was loaded onto a glutathione-Sepharose column (GE Healthcare).

    Techniques: Transformation Assay, Purification

    Pull-down assays from heart lysates. Glutathione Sepharose beads pre-bound with various GST-mXinα fragments were used to pull down associated proteins from total heart lysates. After washing the beads, the bound proteins were eluted from beads

    Journal: Archives of biochemistry and biophysics

    Article Title: Intercalated Disc Protein, mXin?, Suppresses p120-Catenin-Induced Branching Phenotype via Its Interactions with p120-Catenin and Cortactin

    doi: 10.1016/j.abb.2012.12.018

    Figure Lengend Snippet: Pull-down assays from heart lysates. Glutathione Sepharose beads pre-bound with various GST-mXinα fragments were used to pull down associated proteins from total heart lysates. After washing the beads, the bound proteins were eluted from beads

    Article Snippet: The GST fusion proteins were affinity-purified by glutathione sepharose column (GE Healthcare, Pistcataway, NJ) according to the manufacturer’s protocols.

    Techniques:

    Recombinant GST-mXinα directly binds to His-p120-catenin. The pull-down assay was performed with glutathione-Sepharose beads to pull down GST-containing proteins and its interacting proteins from a mixture of purified His-p120-catenin (90 nM)

    Journal: Archives of biochemistry and biophysics

    Article Title: Intercalated Disc Protein, mXin?, Suppresses p120-Catenin-Induced Branching Phenotype via Its Interactions with p120-Catenin and Cortactin

    doi: 10.1016/j.abb.2012.12.018

    Figure Lengend Snippet: Recombinant GST-mXinα directly binds to His-p120-catenin. The pull-down assay was performed with glutathione-Sepharose beads to pull down GST-containing proteins and its interacting proteins from a mixture of purified His-p120-catenin (90 nM)

    Article Snippet: The GST fusion proteins were affinity-purified by glutathione sepharose column (GE Healthcare, Pistcataway, NJ) according to the manufacturer’s protocols.

    Techniques: Recombinant, Pull Down Assay, Purification