Structured Review

Nacalai cosmogel his accept column
SDS-PAGE analysis of the protein fractions collected at each step of purification of HBx(101−136)-(GS) 5 -Bcl-x L . Lane 1, molecular weight marker; lane 2, supernatant; lane 3, His 6 -HBx(101−136)-(GS) 5 -Bcl-x L purified by a <t>COSMOGEL</t> His-accept column; lane 4, His 6 -HBx(101−136)-(GS) 5 -Bcl-x L after dialysis; lane 5, HBx(101−136)-(GS) 5 -Bcl-x L after cleavage of the His 6 -tag by thrombin; lane 6, HBx(101−136)-(GS) 5 -Bcl-x L purified by a Resource Q anion exchange column; lanes 7–9, purified HBx(101−136)-(GS) 5 -Bcl-x L (1-, 3- and 5-µg loadings, respectively).
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1) Product Images from "Expression, purification and characterization of hepatitis B virus X protein BH3-like motif-linker-Bcl-xL fusion protein for structural studies"

Article Title: Expression, purification and characterization of hepatitis B virus X protein BH3-like motif-linker-Bcl-xL fusion protein for structural studies

Journal: Biochemistry and Biophysics Reports

doi: 10.1016/j.bbrep.2016.12.006

SDS-PAGE analysis of the protein fractions collected at each step of purification of HBx(101−136)-(GS) 5 -Bcl-x L . Lane 1, molecular weight marker; lane 2, supernatant; lane 3, His 6 -HBx(101−136)-(GS) 5 -Bcl-x L purified by a COSMOGEL His-accept column; lane 4, His 6 -HBx(101−136)-(GS) 5 -Bcl-x L after dialysis; lane 5, HBx(101−136)-(GS) 5 -Bcl-x L after cleavage of the His 6 -tag by thrombin; lane 6, HBx(101−136)-(GS) 5 -Bcl-x L purified by a Resource Q anion exchange column; lanes 7–9, purified HBx(101−136)-(GS) 5 -Bcl-x L (1-, 3- and 5-µg loadings, respectively).
Figure Legend Snippet: SDS-PAGE analysis of the protein fractions collected at each step of purification of HBx(101−136)-(GS) 5 -Bcl-x L . Lane 1, molecular weight marker; lane 2, supernatant; lane 3, His 6 -HBx(101−136)-(GS) 5 -Bcl-x L purified by a COSMOGEL His-accept column; lane 4, His 6 -HBx(101−136)-(GS) 5 -Bcl-x L after dialysis; lane 5, HBx(101−136)-(GS) 5 -Bcl-x L after cleavage of the His 6 -tag by thrombin; lane 6, HBx(101−136)-(GS) 5 -Bcl-x L purified by a Resource Q anion exchange column; lanes 7–9, purified HBx(101−136)-(GS) 5 -Bcl-x L (1-, 3- and 5-µg loadings, respectively).

Techniques Used: SDS Page, Purification, Molecular Weight, Marker

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Clone Assay:

Article Title: NMR Analyses of the Interaction between the FYVE Domain of Early Endosome Antigen 1 (EEA1) and Phosphoinositide Embedded in a Lipid Bilayer *
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Article Title: Calcium signalling regulates the functions of the bZIP protein VIP1 in touch responses in Arabidopsis thaliana
Article Snippet: The PCR products were digested with Bam HI and Xho I, and cloned into the Bam HI– Xho I site of pRSETB (Thermo Fisher Scientific), generating pRSET-CAM1. .. His-CAM1 in the crude protein sample was bound to COSMOGEL His-Accept (Nacalai Tesque) according to the manufacturer’s instructions, and eluted with 200 m m imidazole.

Article Title: Scaffold Function of Ca2+-Dependent Protein Kinase: Tobacco Ca2+-DEPENDENT PROTEIN KINASE1 Transfers 14-3-3 to the Substrate REPRESSION OF SHOOT GROWTH after Phosphorylation 1-DEPENDENT PROTEIN KINASE1 Transfers 14-3-3 to the Substrate REPRESSION OF SHOOT GROWTH after Phosphorylation 1 [C]-DEPENDENT PROTEIN KINASE1 Transfers 14-3-3 to the Substrate REPRESSION OF SHOOT GROWTH after Phosphoryl
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Centrifugation:

Article Title: Expression, purification and crystallization of a fungal type III polyketide synthase that produces the csypyrones
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Article Title: Metagenomic Analysis of the Sponge Discodermia Reveals the Production of the Cyanobacterial Natural Product Kasumigamide by ‘Entotheonella’
Article Snippet: For MakasA-A1, MakasB-A1, and MakasC-A2, the cells were collected by centrifugation at 10,000 g and resuspended in 50 mM Tris-HCl buffer (pH 8.0), containing 300 mM NaCl, 10% (v/v) glycerol and 5 mM imidazole (buffer A). .. The supernatant was loaded onto a COSMOGEL His-Accept (Nacalai Tesque) column equilibrated with buffer A.

Article Title: Calcium signalling regulates the functions of the bZIP protein VIP1 in touch responses in Arabidopsis thaliana
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Article Title: Characterization of KfrA proteins encoded by a plasmid of Paenibacillus popilliae ATCC 14706T
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Article Title: Hexameric and pentameric complexes of the ExbBD energizer in the Ton system
Article Snippet: The insoluble membrane fraction was removed by centrifugation at 125,000 g for 60 min at 4°C. .. The supernatant was then loaded onto a column filled with a metal affinity resin COSMOGEL His-Accept (Nacalai tesque) at 4°C.

Article Title: Protein phosphatase 2A regulates the nuclear accumulation of the Arabidopsis bZIP protein VIP1 under hypo-osmotic stress
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Amplification:

Article Title: Expression, purification and crystallization of a fungal type III polyketide synthase that produces the csypyrones
Article Snippet: The amplified DNA fragment was digested with Nde I/ Not I and ligated into the Nde I/ Not I sites of the pET-28a vector (Merck Millipore) for expression as a fusion protein with a His6 tag at the N-terminus. .. The supernatant was loaded onto COSMOGEL His-Accept (Nacalai Tesque) equilibrated with buffer A .

Article Title: Metagenomic Analysis of the Sponge Discodermia Reveals the Production of the Cyanobacterial Natural Product Kasumigamide by ‘Entotheonella’
Article Snippet: The makasA-A1 , makasB-A1 , makasB-A2 , makasC-A1 , and makasC-A2 sequences were amplified with the MakasA-A1F/MakasA-A1R, MakasB-A1F/MakasB-A1R, MakasB-A2F/MakasB-A2R, MakasC-A1F/MakasC-A1R, and MakasC-A2F/MakasC-A2R primers, respectively ( ). .. The supernatant was loaded onto a COSMOGEL His-Accept (Nacalai Tesque) column equilibrated with buffer A.

Article Title: Calcium signalling regulates the functions of the bZIP protein VIP1 in touch responses in Arabidopsis thaliana
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Article Title: Scaffold Function of Ca2+-Dependent Protein Kinase: Tobacco Ca2+-DEPENDENT PROTEIN KINASE1 Transfers 14-3-3 to the Substrate REPRESSION OF SHOOT GROWTH after Phosphorylation 1-DEPENDENT PROTEIN KINASE1 Transfers 14-3-3 to the Substrate REPRESSION OF SHOOT GROWTH after Phosphorylation 1 [C]-DEPENDENT PROTEIN KINASE1 Transfers 14-3-3 to the Substrate REPRESSION OF SHOOT GROWTH after Phosphoryl
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Article Title: Protein phosphatase 2A regulates the nuclear accumulation of the Arabidopsis bZIP protein VIP1 under hypo-osmotic stress
Article Snippet: In vitro pull-down assays The CDSs of ATB''δ and FASS were amplified by PCR using the primers listed in Supplementary Table S2 and the cDNA sample prepared as described above. .. The His-tagged proteins in the resulting solutions were bound to COSMOGEL His-Accept resin (Nacalai Tesque, Inc., Kyoto, Japan) according to the manufacturer’s instructions.

Filtration:

Article Title: NMR Analyses of the Interaction between the FYVE Domain of Early Endosome Antigen 1 (EEA1) and Phosphoinositide Embedded in a Lipid Bilayer *
Article Snippet: .. The MSP was purified by Ni2+ affinity chromatography using COSMOGEL His-Accept (Nacalai tesque), followed by the cleavage of the GB1 and hexahistidine tag by HRV-3C protease, and then gel filtration chromatography on a HiLoad 16/60 Superdex 75 pg column (GE Healthcare). ..

Binding Assay:

Article Title: Structural basis for the recognition of the scaffold protein Frmpd4/Preso1 by the TPR domain of the adaptor protein LGN
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Construct:

Article Title: Calcium signalling regulates the functions of the bZIP protein VIP1 in touch responses in Arabidopsis thaliana
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Article Title: Scaffold Function of Ca2+-Dependent Protein Kinase: Tobacco Ca2+-DEPENDENT PROTEIN KINASE1 Transfers 14-3-3 to the Substrate REPRESSION OF SHOOT GROWTH after Phosphorylation 1-DEPENDENT PROTEIN KINASE1 Transfers 14-3-3 to the Substrate REPRESSION OF SHOOT GROWTH after Phosphorylation 1 [C]-DEPENDENT PROTEIN KINASE1 Transfers 14-3-3 to the Substrate REPRESSION OF SHOOT GROWTH after Phosphoryl
Article Snippet: In order to construct His-14-3-3, the complementary DNA fragments encoding 14-3-3 were amplified by PCR with specific primers ( ) and cloned into pET16b (Novagen). .. His-14-3-3 was expressed in E . coli and purified by COSMOGEL His-Accept (Nacalai Tesque).

Incubation:

Article Title: Structural basis for the recognition of the scaffold protein Frmpd4/Preso1 by the TPR domain of the adaptor protein LGN
Article Snippet: Bacterially expressed MBP–Frmpd4-L–His was purified with COSMOGEL His-Accept (Nacalai Tesque) as described previously (Yuzawa et al. , 2011 ). .. LGN-N (0.25 µ M ) and MBP–Frmpd4-L–His or MBP alone (0.5 µ M ) were incubated for 15 min at 277 K with dithiothreitol (5.0 m M ) or H2 O2 (0.5 or 1.0 m M ) in 250 µl of a buffer consisting of 150 m M NaCl, 0.002% Triton X-100, 20 m M HEPES pH 7.4.

Article Title: Expression, purification and crystallization of a fungal type III polyketide synthase that produces the csypyrones
Article Snippet: The cells harbouring the plasmid were cultured to an OD600 of 0.6 in LB medium containing 50 µg ml−1 kanamycin at 310 K. Isopropyl β- d -1-thiogalactopyranoside was then added to a final concentration of 0.5 m M to induce gene expression and the culture was incubated for a further 16 h at 293 K. All of the following procedures were performed at 277 K. The E. coli cells were harvested by centrifugation at 5000 g and resuspended in 50 m M Tris–HCl buffer pH 7.5 containing 250 m M NaCl, 5%( v / v ) glycerol, 5 m M imidazole (buffer A ). .. The supernatant was loaded onto COSMOGEL His-Accept (Nacalai Tesque) equilibrated with buffer A .

Article Title: Calcium signalling regulates the functions of the bZIP protein VIP1 in touch responses in Arabidopsis thaliana
Article Snippet: The resulting solution was mixed with recombinant DNase I (Takara Bio Inc., Kusatsu, Japan), incubated at room temperature until the solution became fluid, and centrifuged at 14 000 g for 20 min. .. His-CAM1 in the crude protein sample was bound to COSMOGEL His-Accept (Nacalai Tesque) according to the manufacturer’s instructions, and eluted with 200 m m imidazole.

Article Title: Characterization of KfrA proteins encoded by a plasmid of Paenibacillus popilliae ATCC 14706T
Article Snippet: .. The retentate was incubated at 30 °C for 2 h, added to 20 μL of 50% slurry of equilibrated COSMOGEL His-Accept (Nacalai Tesque, Kyoto, Japan), and mixed with mild rotation at 4 °C overnight. ..

Expressing:

Article Title: NMR Analyses of the Interaction between the FYVE Domain of Early Endosome Antigen 1 (EEA1) and Phosphoinositide Embedded in a Lipid Bilayer *
Article Snippet: Paragraph title: Protein Expression and Purification ... The MSP was purified by Ni2+ affinity chromatography using COSMOGEL His-Accept (Nacalai tesque), followed by the cleavage of the GB1 and hexahistidine tag by HRV-3C protease, and then gel filtration chromatography on a HiLoad 16/60 Superdex 75 pg column (GE Healthcare).

Article Title: Expression, purification and crystallization of a fungal type III polyketide synthase that produces the csypyrones
Article Snippet: Paragraph title: 2.1. Expression and purification   ... The supernatant was loaded onto COSMOGEL His-Accept (Nacalai Tesque) equilibrated with buffer A .

Article Title: Metagenomic Analysis of the Sponge Discodermia Reveals the Production of the Cyanobacterial Natural Product Kasumigamide by ‘Entotheonella’
Article Snippet: Paragraph title: Heterologous expression of adenylation domains ... The supernatant was loaded onto a COSMOGEL His-Accept (Nacalai Tesque) column equilibrated with buffer A.

Transformation Assay:

Article Title: Expression, purification and crystallization of a fungal type III polyketide synthase that produces the csypyrones
Article Snippet: After confirmation of the sequence, the pET-28a vector encoding full-length CsyB was transformed into Escherichia coli BLR (DE3). .. The supernatant was loaded onto COSMOGEL His-Accept (Nacalai Tesque) equilibrated with buffer A .

Article Title: Calcium signalling regulates the functions of the bZIP protein VIP1 in touch responses in Arabidopsis thaliana
Article Snippet: The transformed E. coli cells were cultured overnight at 37 °C in Luria-Bertani medium with 0.2 m m isopropyl-β- d -thiogalactopyranoside, harvested by centrifugation at 14 000 g for 30 s, resuspended in TBS with 1 mg mL–1 lysozyme (Wako), frozen in liquid nitrogen and thawed at room temperature. .. His-CAM1 in the crude protein sample was bound to COSMOGEL His-Accept (Nacalai Tesque) according to the manufacturer’s instructions, and eluted with 200 m m imidazole.

Article Title: Protein phosphatase 2A regulates the nuclear accumulation of the Arabidopsis bZIP protein VIP1 under hypo-osmotic stress
Article Snippet: The transformed E. coli cells were cultured overnight at 37 °C in LB medium with 0.2 mM IPTG, and harvested by centrifugation at 14 000 g for 30 s. The precipitated cells were frozen and thawed three times in TBS containing 1.5 mg ml–1 lysozyme as described above. .. The His-tagged proteins in the resulting solutions were bound to COSMOGEL His-Accept resin (Nacalai Tesque, Inc., Kyoto, Japan) according to the manufacturer’s instructions.

Derivative Assay:

Article Title: NMR Analyses of the Interaction between the FYVE Domain of Early Endosome Antigen 1 (EEA1) and Phosphoinositide Embedded in a Lipid Bilayer *
Article Snippet: Briefly, the fragment encoding MSP derived from human apoA-I (residues 56–243) was cloned into pGBHPS ( ) and expressed at 28 °C using the Escherichia coli BL21(DE3) strain as a fusion protein with GB1 and hexahistidine tag at the N terminus followed by the HRV-3C protease recognition site. .. The MSP was purified by Ni2+ affinity chromatography using COSMOGEL His-Accept (Nacalai tesque), followed by the cleavage of the GB1 and hexahistidine tag by HRV-3C protease, and then gel filtration chromatography on a HiLoad 16/60 Superdex 75 pg column (GE Healthcare).

Flow Cytometry:

Article Title: Characterization of KfrA proteins encoded by a plasmid of Paenibacillus popilliae ATCC 14706T
Article Snippet: The retentate was incubated at 30 °C for 2 h, added to 20 μL of 50% slurry of equilibrated COSMOGEL His-Accept (Nacalai Tesque, Kyoto, Japan), and mixed with mild rotation at 4 °C overnight. .. After centrifugation (20 °C, 1 min, 3000 × g ), the flow-through fraction was collected and washed five times with 500 μL of 20 mM NaH2 PO4 –Na2 HPO4 buffer (pH7.4), 0.5 M NaCl, and 5 mM imidazole.

Chromatography:

Article Title: NMR Analyses of the Interaction between the FYVE Domain of Early Endosome Antigen 1 (EEA1) and Phosphoinositide Embedded in a Lipid Bilayer *
Article Snippet: .. The MSP was purified by Ni2+ affinity chromatography using COSMOGEL His-Accept (Nacalai tesque), followed by the cleavage of the GB1 and hexahistidine tag by HRV-3C protease, and then gel filtration chromatography on a HiLoad 16/60 Superdex 75 pg column (GE Healthcare). ..

Article Title: Expression, purification and crystallization of a fungal type III polyketide synthase that produces the csypyrones
Article Snippet: The supernatant was loaded onto COSMOGEL His-Accept (Nacalai Tesque) equilibrated with buffer A . .. The protein solution was concentrated to 5 ml, further purified to homogeneity by gel-filtration chromatography on HiLoad 16/60 Superdex 200 pg (GE Healthcare) and concentrated to 13 mg ml−1 in 20 m M Tris–HCl buffer pH 7.5 containing 150 m M NaCl, 2 m M DTT.

Protease Inhibitor:

Article Title: Hexameric and pentameric complexes of the ExbBD energizer in the Ton system
Article Snippet: The pellet was resuspended in 50 mM Tris-HCl, pH 8.0, 0.3 M NaCl, 10% (v/v) glycerol, 10 mM imidazole, protease inhibitor cocktail cOmplete (Roche), DNase I (Roche), 5 mM MgCl2 , and 0.2 mg/ml lysozyme and disrupted with ~7 passages at ~10,000 psi through a high pressure homogenizer EmulsiFlex C-5 (AVESTIN). .. The supernatant was then loaded onto a column filled with a metal affinity resin COSMOGEL His-Accept (Nacalai tesque) at 4°C.

Transferring:

Article Title: Characterization of KfrA proteins encoded by a plasmid of Paenibacillus popilliae ATCC 14706T
Article Snippet: Multimer formation was initiated by transferring to 20 mM NaH2 PO4 –Na2 HPO4 (pH 7.4), and 137 mM NaCl using a 10 K Nanosep device. .. The retentate was incubated at 30 °C for 2 h, added to 20 μL of 50% slurry of equilibrated COSMOGEL His-Accept (Nacalai Tesque, Kyoto, Japan), and mixed with mild rotation at 4 °C overnight.

Cell Culture:

Article Title: Expression, purification and crystallization of a fungal type III polyketide synthase that produces the csypyrones
Article Snippet: The cells harbouring the plasmid were cultured to an OD600 of 0.6 in LB medium containing 50 µg ml−1 kanamycin at 310 K. Isopropyl β- d -1-thiogalactopyranoside was then added to a final concentration of 0.5 m M to induce gene expression and the culture was incubated for a further 16 h at 293 K. All of the following procedures were performed at 277 K. The E. coli cells were harvested by centrifugation at 5000 g and resuspended in 50 m M Tris–HCl buffer pH 7.5 containing 250 m M NaCl, 5%( v / v ) glycerol, 5 m M imidazole (buffer A ). .. The supernatant was loaded onto COSMOGEL His-Accept (Nacalai Tesque) equilibrated with buffer A .

Article Title: Metagenomic Analysis of the Sponge Discodermia Reveals the Production of the Cyanobacterial Natural Product Kasumigamide by ‘Entotheonella’
Article Snippet: The E . coli BLR(DE3) cells harboring the plasmids pCold II makasA-A1 /makasB-A1 /makasC-A2 and NhisMBP-pET28b(+) makasB-A2 /makasC-A1 were cultured at 37°C to an OD600 of 0.6–0.8, in LB medium, containing 100 μg/ml ampicillin or 50 μg/ml kanamycin. .. The supernatant was loaded onto a COSMOGEL His-Accept (Nacalai Tesque) column equilibrated with buffer A.

Article Title: Calcium signalling regulates the functions of the bZIP protein VIP1 in touch responses in Arabidopsis thaliana
Article Snippet: The transformed E. coli cells were cultured overnight at 37 °C in Luria-Bertani medium with 0.2 m m isopropyl-β- d -thiogalactopyranoside, harvested by centrifugation at 14 000 g for 30 s, resuspended in TBS with 1 mg mL–1 lysozyme (Wako), frozen in liquid nitrogen and thawed at room temperature. .. His-CAM1 in the crude protein sample was bound to COSMOGEL His-Accept (Nacalai Tesque) according to the manufacturer’s instructions, and eluted with 200 m m imidazole.

Article Title: Protein phosphatase 2A regulates the nuclear accumulation of the Arabidopsis bZIP protein VIP1 under hypo-osmotic stress
Article Snippet: The transformed E. coli cells were cultured overnight at 37 °C in LB medium with 0.2 mM IPTG, and harvested by centrifugation at 14 000 g for 30 s. The precipitated cells were frozen and thawed three times in TBS containing 1.5 mg ml–1 lysozyme as described above. .. The His-tagged proteins in the resulting solutions were bound to COSMOGEL His-Accept resin (Nacalai Tesque, Inc., Kyoto, Japan) according to the manufacturer’s instructions.

Sequencing:

Article Title: Expression, purification and crystallization of a fungal type III polyketide synthase that produces the csypyrones
Article Snippet: After confirmation of the sequence, the pET-28a vector encoding full-length CsyB was transformed into Escherichia coli BLR (DE3). .. The supernatant was loaded onto COSMOGEL His-Accept (Nacalai Tesque) equilibrated with buffer A .

Article Title: Calcium signalling regulates the functions of the bZIP protein VIP1 in touch responses in Arabidopsis thaliana
Article Snippet: Using this solution as the template, the primer pair 5′-CCT GGATCCATGG CGGATCAACTCACTGACGAAC-3′ and 5′-CAC CTCGAG TCACTTCATCATAATCTTGAC-3′ ( Nco I, Bam HI and Xho I sites are underlined), and the KOD FX Neo DNA polymerase (Toyobo), the coding sequence of CAM1 was amplified by PCR. .. His-CAM1 in the crude protein sample was bound to COSMOGEL His-Accept (Nacalai Tesque) according to the manufacturer’s instructions, and eluted with 200 m m imidazole.

Recombinant:

Article Title: Expression, purification and crystallization of a fungal type III polyketide synthase that produces the csypyrones
Article Snippet: The supernatant was loaded onto COSMOGEL His-Accept (Nacalai Tesque) equilibrated with buffer A . .. After washing the resin with buffer A containing 10 m M imidazole, the recombinant CsyB protein was subsequently eluted with buffer A containing 300 m M imidazole.

Article Title: Calcium signalling regulates the functions of the bZIP protein VIP1 in touch responses in Arabidopsis thaliana
Article Snippet: The resulting solution was mixed with recombinant DNase I (Takara Bio Inc., Kusatsu, Japan), incubated at room temperature until the solution became fluid, and centrifuged at 14 000 g for 20 min. .. His-CAM1 in the crude protein sample was bound to COSMOGEL His-Accept (Nacalai Tesque) according to the manufacturer’s instructions, and eluted with 200 m m imidazole.

Article Title: Characterization of KfrA proteins encoded by a plasmid of Paenibacillus popilliae ATCC 14706T
Article Snippet: Paragraph title: Molecular interaction between recombinant Orf8 and Orf16 ... The retentate was incubated at 30 °C for 2 h, added to 20 μL of 50% slurry of equilibrated COSMOGEL His-Accept (Nacalai Tesque, Kyoto, Japan), and mixed with mild rotation at 4 °C overnight.

Article Title: Scaffold Function of Ca2+-Dependent Protein Kinase: Tobacco Ca2+-DEPENDENT PROTEIN KINASE1 Transfers 14-3-3 to the Substrate REPRESSION OF SHOOT GROWTH after Phosphorylation 1-DEPENDENT PROTEIN KINASE1 Transfers 14-3-3 to the Substrate REPRESSION OF SHOOT GROWTH after Phosphorylation 1 [C]-DEPENDENT PROTEIN KINASE1 Transfers 14-3-3 to the Substrate REPRESSION OF SHOOT GROWTH after Phosphoryl
Article Snippet: Paragraph title: Preparation of Recombinant Proteins ... His-14-3-3 was expressed in E . coli and purified by COSMOGEL His-Accept (Nacalai Tesque).

Molecular Weight:

Article Title: Hexameric and pentameric complexes of the ExbBD energizer in the Ton system
Article Snippet: The supernatant was then loaded onto a column filled with a metal affinity resin COSMOGEL His-Accept (Nacalai tesque) at 4°C. .. Fractions eluting at ~0.3 M imidazole were collected and concentrated using a spin concentrator with a molecular weight cut-off of 100,000.

Size-exclusion Chromatography:

Article Title: Hexameric and pentameric complexes of the ExbBD energizer in the Ton system
Article Snippet: The ExbBD complex was purified by immobilized metal affinity (IMAC) and size-exclusion chromatography (SEC). .. The supernatant was then loaded onto a column filled with a metal affinity resin COSMOGEL His-Accept (Nacalai tesque) at 4°C.

Purification:

Article Title: Structural basis for the recognition of the scaffold protein Frmpd4/Preso1 by the TPR domain of the adaptor protein LGN
Article Snippet: .. Bacterially expressed MBP–Frmpd4-L–His was purified with COSMOGEL His-Accept (Nacalai Tesque) as described previously (Yuzawa et al. , 2011 ). .. LGN-N (0.25 µ M ) and MBP–Frmpd4-L–His or MBP alone (0.5 µ M ) were incubated for 15 min at 277 K with dithiothreitol (5.0 m M ) or H2 O2 (0.5 or 1.0 m M ) in 250 µl of a buffer consisting of 150 m M NaCl, 0.002% Triton X-100, 20 m M HEPES pH 7.4.

Article Title: NMR Analyses of the Interaction between the FYVE Domain of Early Endosome Antigen 1 (EEA1) and Phosphoinositide Embedded in a Lipid Bilayer *
Article Snippet: .. The MSP was purified by Ni2+ affinity chromatography using COSMOGEL His-Accept (Nacalai tesque), followed by the cleavage of the GB1 and hexahistidine tag by HRV-3C protease, and then gel filtration chromatography on a HiLoad 16/60 Superdex 75 pg column (GE Healthcare). ..

Article Title: Expression, purification and crystallization of a fungal type III polyketide synthase that produces the csypyrones
Article Snippet: Paragraph title: 2.1. Expression and purification   ... The supernatant was loaded onto COSMOGEL His-Accept (Nacalai Tesque) equilibrated with buffer A .

Article Title: Calcium signalling regulates the functions of the bZIP protein VIP1 in touch responses in Arabidopsis thaliana
Article Snippet: His-CAM1 in the crude protein sample was bound to COSMOGEL His-Accept (Nacalai Tesque) according to the manufacturer’s instructions, and eluted with 200 m m imidazole. .. The resulting solution was used as a solution containing purified His-CAM1.

Article Title: Scaffold Function of Ca2+-Dependent Protein Kinase: Tobacco Ca2+-DEPENDENT PROTEIN KINASE1 Transfers 14-3-3 to the Substrate REPRESSION OF SHOOT GROWTH after Phosphorylation 1-DEPENDENT PROTEIN KINASE1 Transfers 14-3-3 to the Substrate REPRESSION OF SHOOT GROWTH after Phosphorylation 1 [C]-DEPENDENT PROTEIN KINASE1 Transfers 14-3-3 to the Substrate REPRESSION OF SHOOT GROWTH after Phosphoryl
Article Snippet: .. His-14-3-3 was expressed in E . coli and purified by COSMOGEL His-Accept (Nacalai Tesque). .. Purified recombinant GST-NtCDPK1 was autophosphorylated in a reaction mixture containing 20 m m Tris-HCl, pH 7.5, 10 m m MgCl2 , 0.5 m m CaCl2 , 0.1% (v/v) Triton X-100, and 0.05% (v/v) β-mercaptoethanol with 1 m m ATP (cold assay) or 1 m m ATP supplemented with [γ-32 P]ATP (5,000 µCi mmol−1 , 10–20 µCi per reaction; hot assay) at 30°C for 30 min. GST-NtCDPK1 was dephosphorylated by λ-protein phosphatase at 30°C for 30 min according to the manufacturer’s manual (New England Biolabs).

Article Title: Hexameric and pentameric complexes of the ExbBD energizer in the Ton system
Article Snippet: Paragraph title: Cloning, co-expression and purification of the ExbBD complex ... The supernatant was then loaded onto a column filled with a metal affinity resin COSMOGEL His-Accept (Nacalai tesque) at 4°C.

Article Title: Protein phosphatase 2A regulates the nuclear accumulation of the Arabidopsis bZIP protein VIP1 under hypo-osmotic stress
Article Snippet: The His-tagged proteins in the resulting solutions were bound to COSMOGEL His-Accept resin (Nacalai Tesque, Inc., Kyoto, Japan) according to the manufacturer’s instructions. .. The suspensions were centrifuged for 3 min at 10 000 g at room temperature, and supernatants were used as the solutions containing purified His-tagged proteins.

Polymerase Chain Reaction:

Article Title: Expression, purification and crystallization of a fungal type III polyketide synthase that produces the csypyrones
Article Snippet: The cDNA encoding full-length CsyB from A. oryzae was purchased from Operon Biotechnologies Inc. and was used as the template to amplify the CsyB gene by PCR using 5′-CCT CATATG ATCGAACCGTTACCGAC-3′ as the sense primer, which introduces an Nde I restriction site, and 5′-AAT GCGGCCGC TTATGCGTGCAGATACGAGC-3′ as the antisense primer, which introduces a Not I restriction site. .. The supernatant was loaded onto COSMOGEL His-Accept (Nacalai Tesque) equilibrated with buffer A .

Article Title: Calcium signalling regulates the functions of the bZIP protein VIP1 in touch responses in Arabidopsis thaliana
Article Snippet: The PCR products were digested with Bam HI and Xho I, and cloned into the Bam HI– Xho I site of pRSETB (Thermo Fisher Scientific), generating pRSET-CAM1. .. His-CAM1 in the crude protein sample was bound to COSMOGEL His-Accept (Nacalai Tesque) according to the manufacturer’s instructions, and eluted with 200 m m imidazole.

Article Title: Scaffold Function of Ca2+-Dependent Protein Kinase: Tobacco Ca2+-DEPENDENT PROTEIN KINASE1 Transfers 14-3-3 to the Substrate REPRESSION OF SHOOT GROWTH after Phosphorylation 1-DEPENDENT PROTEIN KINASE1 Transfers 14-3-3 to the Substrate REPRESSION OF SHOOT GROWTH after Phosphorylation 1 [C]-DEPENDENT PROTEIN KINASE1 Transfers 14-3-3 to the Substrate REPRESSION OF SHOOT GROWTH after Phosphoryl
Article Snippet: In order to construct His-14-3-3, the complementary DNA fragments encoding 14-3-3 were amplified by PCR with specific primers ( ) and cloned into pET16b (Novagen). .. His-14-3-3 was expressed in E . coli and purified by COSMOGEL His-Accept (Nacalai Tesque).

Article Title: Protein phosphatase 2A regulates the nuclear accumulation of the Arabidopsis bZIP protein VIP1 under hypo-osmotic stress
Article Snippet: The PCR product with ATB''δ was digested with Spe I and Xho I, and that with FASS was digested with Spe I and Sal I. .. The His-tagged proteins in the resulting solutions were bound to COSMOGEL His-Accept resin (Nacalai Tesque, Inc., Kyoto, Japan) according to the manufacturer’s instructions.

Affinity Chromatography:

Article Title: NMR Analyses of the Interaction between the FYVE Domain of Early Endosome Antigen 1 (EEA1) and Phosphoinositide Embedded in a Lipid Bilayer *
Article Snippet: .. The MSP was purified by Ni2+ affinity chromatography using COSMOGEL His-Accept (Nacalai tesque), followed by the cleavage of the GB1 and hexahistidine tag by HRV-3C protease, and then gel filtration chromatography on a HiLoad 16/60 Superdex 75 pg column (GE Healthcare). ..

SDS Page:

Article Title: Structural basis for the recognition of the scaffold protein Frmpd4/Preso1 by the TPR domain of the adaptor protein LGN
Article Snippet: Bacterially expressed MBP–Frmpd4-L–His was purified with COSMOGEL His-Accept (Nacalai Tesque) as described previously (Yuzawa et al. , 2011 ). .. Proteins were pulled down with Amylose Resin and subjected to SDS–PAGE followed by staining with Coomassie Brilliant Blue (CBB).

Article Title: Characterization of KfrA proteins encoded by a plasmid of Paenibacillus popilliae ATCC 14706T
Article Snippet: The retentate was incubated at 30 °C for 2 h, added to 20 μL of 50% slurry of equilibrated COSMOGEL His-Accept (Nacalai Tesque, Kyoto, Japan), and mixed with mild rotation at 4 °C overnight. .. The proteins in the flow-through and eluent fractions were separated by 10% SDS-PAGE.

Plasmid Preparation:

Article Title: Expression, purification and crystallization of a fungal type III polyketide synthase that produces the csypyrones
Article Snippet: The cells harbouring the plasmid were cultured to an OD600 of 0.6 in LB medium containing 50 µg ml−1 kanamycin at 310 K. Isopropyl β- d -1-thiogalactopyranoside was then added to a final concentration of 0.5 m M to induce gene expression and the culture was incubated for a further 16 h at 293 K. All of the following procedures were performed at 277 K. The E. coli cells were harvested by centrifugation at 5000 g and resuspended in 50 m M Tris–HCl buffer pH 7.5 containing 250 m M NaCl, 5%( v / v ) glycerol, 5 m M imidazole (buffer A ). .. The supernatant was loaded onto COSMOGEL His-Accept (Nacalai Tesque) equilibrated with buffer A .

Article Title: Protein phosphatase 2A regulates the nuclear accumulation of the Arabidopsis bZIP protein VIP1 under hypo-osmotic stress
Article Snippet: These DNA fragments were inserted into the Nhe I–Xho I site of the pRSETB vector (Thermo Fisher Scientific), generating pRSET-ATB''δ and pRSET-FASS. .. The His-tagged proteins in the resulting solutions were bound to COSMOGEL His-Accept resin (Nacalai Tesque, Inc., Kyoto, Japan) according to the manufacturer’s instructions.

Positron Emission Tomography:

Article Title: Expression, purification and crystallization of a fungal type III polyketide synthase that produces the csypyrones
Article Snippet: After confirmation of the sequence, the pET-28a vector encoding full-length CsyB was transformed into Escherichia coli BLR (DE3). .. The supernatant was loaded onto COSMOGEL His-Accept (Nacalai Tesque) equilibrated with buffer A .

In Vitro:

Article Title: Calcium signalling regulates the functions of the bZIP protein VIP1 in touch responses in Arabidopsis thaliana
Article Snippet: Paragraph title: In vitro protein–protein interaction analyses ... His-CAM1 in the crude protein sample was bound to COSMOGEL His-Accept (Nacalai Tesque) according to the manufacturer’s instructions, and eluted with 200 m m imidazole.

Article Title: Protein phosphatase 2A regulates the nuclear accumulation of the Arabidopsis bZIP protein VIP1 under hypo-osmotic stress
Article Snippet: Paragraph title: In vitro pull-down assays ... The His-tagged proteins in the resulting solutions were bound to COSMOGEL His-Accept resin (Nacalai Tesque, Inc., Kyoto, Japan) according to the manufacturer’s instructions.

Concentration Assay:

Article Title: Expression, purification and crystallization of a fungal type III polyketide synthase that produces the csypyrones
Article Snippet: The cells harbouring the plasmid were cultured to an OD600 of 0.6 in LB medium containing 50 µg ml−1 kanamycin at 310 K. Isopropyl β- d -1-thiogalactopyranoside was then added to a final concentration of 0.5 m M to induce gene expression and the culture was incubated for a further 16 h at 293 K. All of the following procedures were performed at 277 K. The E. coli cells were harvested by centrifugation at 5000 g and resuspended in 50 m M Tris–HCl buffer pH 7.5 containing 250 m M NaCl, 5%( v / v ) glycerol, 5 m M imidazole (buffer A ). .. The supernatant was loaded onto COSMOGEL His-Accept (Nacalai Tesque) equilibrated with buffer A .

Article Title: Hexameric and pentameric complexes of the ExbBD energizer in the Ton system
Article Snippet: The supernatant was then loaded onto a column filled with a metal affinity resin COSMOGEL His-Accept (Nacalai tesque) at 4°C. .. The column was washed with the solubilization buffer except for containing 0.018% (w/v; × 2 critical micelle concentration; cmc) DDM, and developed with a stepwise gradient of imidazole concentration.

Article Title: Protein phosphatase 2A regulates the nuclear accumulation of the Arabidopsis bZIP protein VIP1 under hypo-osmotic stress
Article Snippet: The His-tagged proteins in the resulting solutions were bound to COSMOGEL His-Accept resin (Nacalai Tesque, Inc., Kyoto, Japan) according to the manufacturer’s instructions. .. When used, CaCl2 and EDTA (pH 8.0) were added to these solutions to obtain a final concentration of 20 mM (for both CaCl2 and EDTA).

Staining:

Article Title: Structural basis for the recognition of the scaffold protein Frmpd4/Preso1 by the TPR domain of the adaptor protein LGN
Article Snippet: Bacterially expressed MBP–Frmpd4-L–His was purified with COSMOGEL His-Accept (Nacalai Tesque) as described previously (Yuzawa et al. , 2011 ). .. Proteins were pulled down with Amylose Resin and subjected to SDS–PAGE followed by staining with Coomassie Brilliant Blue (CBB).

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  • 84
    Nacalai cosmogel his accept column
    SDS-PAGE analysis of the protein fractions collected at each step of purification of HBx(101−136)-(GS) 5 -Bcl-x L . Lane 1, molecular weight marker; lane 2, supernatant; lane 3, His 6 -HBx(101−136)-(GS) 5 -Bcl-x L purified by a <t>COSMOGEL</t> His-accept column; lane 4, His 6 -HBx(101−136)-(GS) 5 -Bcl-x L after dialysis; lane 5, HBx(101−136)-(GS) 5 -Bcl-x L after cleavage of the His 6 -tag by thrombin; lane 6, HBx(101−136)-(GS) 5 -Bcl-x L purified by a Resource Q anion exchange column; lanes 7–9, purified HBx(101−136)-(GS) 5 -Bcl-x L (1-, 3- and 5-µg loadings, respectively).
    Cosmogel His Accept Column, supplied by Nacalai, used in various techniques. Bioz Stars score: 84/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cosmogel his accept column/product/Nacalai
    Average 84 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    cosmogel his accept column - by Bioz Stars, 2020-01
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    89
    Nacalai ni2 sepharose resin
    SDS-PAGE analysis of the protein fractions collected at each step of purification of HBx(101−136)-(GS) 5 -Bcl-x L . Lane 1, molecular weight marker; lane 2, supernatant; lane 3, His 6 -HBx(101−136)-(GS) 5 -Bcl-x L purified by a <t>COSMOGEL</t> His-accept column; lane 4, His 6 -HBx(101−136)-(GS) 5 -Bcl-x L after dialysis; lane 5, HBx(101−136)-(GS) 5 -Bcl-x L after cleavage of the His 6 -tag by thrombin; lane 6, HBx(101−136)-(GS) 5 -Bcl-x L purified by a Resource Q anion exchange column; lanes 7–9, purified HBx(101−136)-(GS) 5 -Bcl-x L (1-, 3- and 5-µg loadings, respectively).
    Ni2 Sepharose Resin, supplied by Nacalai, used in various techniques. Bioz Stars score: 89/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ni2 sepharose resin/product/Nacalai
    Average 89 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    ni2 sepharose resin - by Bioz Stars, 2020-01
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    94
    Nacalai ni agarose
    SDS-PAGE analysis of the protein fractions collected at each step of purification of HBx(101−136)-(GS) 5 -Bcl-x L . Lane 1, molecular weight marker; lane 2, supernatant; lane 3, His 6 -HBx(101−136)-(GS) 5 -Bcl-x L purified by a <t>COSMOGEL</t> His-accept column; lane 4, His 6 -HBx(101−136)-(GS) 5 -Bcl-x L after dialysis; lane 5, HBx(101−136)-(GS) 5 -Bcl-x L after cleavage of the His 6 -tag by thrombin; lane 6, HBx(101−136)-(GS) 5 -Bcl-x L purified by a Resource Q anion exchange column; lanes 7–9, purified HBx(101−136)-(GS) 5 -Bcl-x L (1-, 3- and 5-µg loadings, respectively).
    Ni Agarose, supplied by Nacalai, used in various techniques. Bioz Stars score: 94/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ni agarose/product/Nacalai
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    Price from $9.99 to $1999.99
    ni agarose - by Bioz Stars, 2020-01
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    SDS-PAGE analysis of the protein fractions collected at each step of purification of HBx(101−136)-(GS) 5 -Bcl-x L . Lane 1, molecular weight marker; lane 2, supernatant; lane 3, His 6 -HBx(101−136)-(GS) 5 -Bcl-x L purified by a COSMOGEL His-accept column; lane 4, His 6 -HBx(101−136)-(GS) 5 -Bcl-x L after dialysis; lane 5, HBx(101−136)-(GS) 5 -Bcl-x L after cleavage of the His 6 -tag by thrombin; lane 6, HBx(101−136)-(GS) 5 -Bcl-x L purified by a Resource Q anion exchange column; lanes 7–9, purified HBx(101−136)-(GS) 5 -Bcl-x L (1-, 3- and 5-µg loadings, respectively).

    Journal: Biochemistry and Biophysics Reports

    Article Title: Expression, purification and characterization of hepatitis B virus X protein BH3-like motif-linker-Bcl-xL fusion protein for structural studies

    doi: 10.1016/j.bbrep.2016.12.006

    Figure Lengend Snippet: SDS-PAGE analysis of the protein fractions collected at each step of purification of HBx(101−136)-(GS) 5 -Bcl-x L . Lane 1, molecular weight marker; lane 2, supernatant; lane 3, His 6 -HBx(101−136)-(GS) 5 -Bcl-x L purified by a COSMOGEL His-accept column; lane 4, His 6 -HBx(101−136)-(GS) 5 -Bcl-x L after dialysis; lane 5, HBx(101−136)-(GS) 5 -Bcl-x L after cleavage of the His 6 -tag by thrombin; lane 6, HBx(101−136)-(GS) 5 -Bcl-x L purified by a Resource Q anion exchange column; lanes 7–9, purified HBx(101−136)-(GS) 5 -Bcl-x L (1-, 3- and 5-µg loadings, respectively).

    Article Snippet: The supernatant containing the His6 -tagged protein, which was obtained by centrifugation, was loaded onto a COSMOGEL His-accept column (3–4 mL bed volume) (Nacalai Tesque Inc.).

    Techniques: SDS Page, Purification, Molecular Weight, Marker