histrap ff crude column  (GE Healthcare)

 
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    HisTrap FF
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    HisTrap FF
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    Structured Review

    GE Healthcare histrap ff crude column
    Purified trKMO. (A) Chromatogram produced during NiNTA affinity purification of truncate KMO. (B) SDS-PAGE protein gel stained with simplyblue showing <t>HisTrap</t> purification of truncated human KMO protein, (a) Cell-free extract prior to affinity chromatography, (b) flowthrough proteins which did not bind to the column, (c) first column wash, (d) second column wash, (e) third column wash, (f) pure truncated human KMO protein (44 kDa) from the fractions under the chromatogram. (C) Cartoon illustrating the trKMO construct. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

    https://www.bioz.com/result/histrap ff crude column/product/GE Healthcare
    Average 99 stars, based on 245 article reviews
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    Images

    1) Product Images from "Bacterial expression of human kynurenine 3-monooxygenase: Solubility, activity, purification"

    Article Title: Bacterial expression of human kynurenine 3-monooxygenase: Solubility, activity, purification

    Journal: Protein Expression and Purification

    doi: 10.1016/j.pep.2013.11.015

    Purified trKMO. (A) Chromatogram produced during NiNTA affinity purification of truncate KMO. (B) SDS-PAGE protein gel stained with simplyblue showing HisTrap purification of truncated human KMO protein, (a) Cell-free extract prior to affinity chromatography, (b) flowthrough proteins which did not bind to the column, (c) first column wash, (d) second column wash, (e) third column wash, (f) pure truncated human KMO protein (44 kDa) from the fractions under the chromatogram. (C) Cartoon illustrating the trKMO construct. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
    Figure Legend Snippet: Purified trKMO. (A) Chromatogram produced during NiNTA affinity purification of truncate KMO. (B) SDS-PAGE protein gel stained with simplyblue showing HisTrap purification of truncated human KMO protein, (a) Cell-free extract prior to affinity chromatography, (b) flowthrough proteins which did not bind to the column, (c) first column wash, (d) second column wash, (e) third column wash, (f) pure truncated human KMO protein (44 kDa) from the fractions under the chromatogram. (C) Cartoon illustrating the trKMO construct. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

    Techniques Used: Purification, Produced, Affinity Purification, SDS Page, Staining, Affinity Chromatography, Construct

    2) Product Images from "Protein expression, characterization, crystallization and preliminary X-ray crystallographic analysis of a Fic protein from Clostridium difficile"

    Article Title: Protein expression, characterization, crystallization and preliminary X-ray crystallographic analysis of a Fic protein from Clostridium difficile

    Journal: Acta Crystallographica. Section F, Structural Biology Communications

    doi: 10.1107/S2053230X1400987X

    Purification and oligomeric state of CdFic SE/AA . ( a ) Coomassie-stained 4–15% gradient SDS–PAGE of purified CdFic SE/AA . Lane M , molecular-weight marker (labelled in kDa). Lane 1, elution from the HisTrap affinity column, 1.8 µg
    Figure Legend Snippet: Purification and oligomeric state of CdFic SE/AA . ( a ) Coomassie-stained 4–15% gradient SDS–PAGE of purified CdFic SE/AA . Lane M , molecular-weight marker (labelled in kDa). Lane 1, elution from the HisTrap affinity column, 1.8 µg

    Techniques Used: Purification, Staining, SDS Page, Molecular Weight, Marker, Affinity Column

    Related Articles

    Centrifugation:

    Article Title: Engineering of monobody conjugates for human EphA2-specific optical imaging
    Article Snippet: .. Supernatants were obtained by centrifugation at 10,000 rpm at 4°C and applied to a HisTrap FF column (GE Healthcare Biosciences, PA) in an AKTA FPLC system (GE Healthcare Biosciences). ..

    Recombinant:

    Article Title: Characterization and Functional Analysis of atl, a Novel Gene Encoding Autolysin in Streptococcus suis
    Article Snippet: .. The Atl fusion protein was extracted from a mid-log culture, which had been induced with 1 mM IPTG for 5 h. The IPTG-inducible expression plasmid pET32a contained a start codon followed by a 6×His tag sequence, allowing purification of the recombinant autolysin by using a HisTrap FF column. .. As shown in , an apparent protein band of approximately 96 kDa appeared in the induced recombinant E. coli , while it was absent in the uninduced recombinant E. coli or induced E. coli containing control plasmid.

    Chromatography:

    Article Title: The purification of recombinant human tyrosinase from insect larvae infected with the baculovirus vector
    Article Snippet: .. T.ni larvae biomass containing the protein of interest (Allotropic Tech., MD) Wet ice 50 ml tubes (Corning, NY) Lysis buffer (see recipe) 1-Phenyl-2-thiourea (PTU; Sigma-Aldrich, MO) DNase I (Thermo Fisher Scientific, PA) Complete set of protease inhibitors (Roche, USA) tris(2-carboxyethyl)phosphine (TCEP; Thermo Scientific, DE) Fisher Science Education pH meter (Thermo Fisher Scientific, MA) Omni Tissue Homogenizer TH (Omni International, GA) Hard Tissue Omni Tip™ Homogenizer Probes (Omni International, GA) Orbitron Rotator (Boekel Scientific, PA) Ultrasonic Processor GE130PB (Hielscher System, Germany) Sorvall Lynx 4000 Centrifuge (Thermo Scientific, DE) Rotor for centrifuge: Fiberlite, F21-8×50y (capacity 8 × 50 ml) Binding buffer (see recipe) Elution buffer (see recipe) Immobilized metal affinity (IMAC) column - HisTrap FF Crude (GE Healthcare, NJ) BioLogic Duo-Flow chromatography system (Bio-Rad, CA) Gel-filtration buffer (see recipe) NanoDrop 2000c UV-Vis spectrophotometer (Thermo Scientific, DE) 4-15% polyacrylamide gels (Bio-Rad, CA) 3,4-Dihydroxy-L-phenylalanine, L-DOPA (Sigma Aldrich, MO) MO) SpectraMax i3 Multi-Mode Microplate Detection Platform (Molecular Devices, CA) Clear-bottom polystyrene 96-well plates (Corning, NY) Dialysis membranes: SnakeSkin dialysis tubing or Slide-A-Lyzer dialysis cassettes, 10K MWCO (ThermoFisher Scientific, MA) Gel-filtration columns: HiPrep 16/60 Sephacryl S-100 and Superdex 75 10/300 (GE HealthCare, NJ) Protein standards (Bio-Rad, CA) Anti-His antibody (Life Technologies, NY) Anti-tyrosinase antibody (T311 Santa Cruz Biotechnology, CA) Centrifuge 5415R (Eppendorf, Germany) UN-SCAN-IT gel ™ gel analysis software (Silk Scientific, Inc., UT) Stirring devices Centrifuge tubes Additional materials and equipment for SDS-PAGE ( UNIT 10.1 ) and Western blot ( UNIT 10.8 ) analysis. .. The DNA of hTyrCtr included residues 19-469 of the human tyrosinase, a TEV-protease cleavage site (NLYFQG sequence), and a 6×His-Tag ( ) was commercially synthesized, expressed in baculovirus and produced in whole insect T. ni larvae (Allotropic Tech., MD).

    Flow Cytometry:

    Article Title: Computational redesign of the Escherichia coli ribose-binding protein ligand binding pocket for 1,3-cyclohexanediol and cyclohexanol
    Article Snippet: .. The clean lysate was next loaded onto a HisTrap HP column (HisTrap FF crude 1 ml, GE Healthcare) at 4 °C and flow rate of 0.5 ml min−1 , followed by washes of, consecutively, 10 column volumes (cv, equal to 1 ml) of buffer A with 20 mM imidazole, 1.5 cv of buffer A with 40 mM imidazole and 1.5 cv of buffer A with 80 mM imidazole. ..

    Spectrophotometry:

    Article Title: The purification of recombinant human tyrosinase from insect larvae infected with the baculovirus vector
    Article Snippet: .. T.ni larvae biomass containing the protein of interest (Allotropic Tech., MD) Wet ice 50 ml tubes (Corning, NY) Lysis buffer (see recipe) 1-Phenyl-2-thiourea (PTU; Sigma-Aldrich, MO) DNase I (Thermo Fisher Scientific, PA) Complete set of protease inhibitors (Roche, USA) tris(2-carboxyethyl)phosphine (TCEP; Thermo Scientific, DE) Fisher Science Education pH meter (Thermo Fisher Scientific, MA) Omni Tissue Homogenizer TH (Omni International, GA) Hard Tissue Omni Tip™ Homogenizer Probes (Omni International, GA) Orbitron Rotator (Boekel Scientific, PA) Ultrasonic Processor GE130PB (Hielscher System, Germany) Sorvall Lynx 4000 Centrifuge (Thermo Scientific, DE) Rotor for centrifuge: Fiberlite, F21-8×50y (capacity 8 × 50 ml) Binding buffer (see recipe) Elution buffer (see recipe) Immobilized metal affinity (IMAC) column - HisTrap FF Crude (GE Healthcare, NJ) BioLogic Duo-Flow chromatography system (Bio-Rad, CA) Gel-filtration buffer (see recipe) NanoDrop 2000c UV-Vis spectrophotometer (Thermo Scientific, DE) 4-15% polyacrylamide gels (Bio-Rad, CA) 3,4-Dihydroxy-L-phenylalanine, L-DOPA (Sigma Aldrich, MO) MO) SpectraMax i3 Multi-Mode Microplate Detection Platform (Molecular Devices, CA) Clear-bottom polystyrene 96-well plates (Corning, NY) Dialysis membranes: SnakeSkin dialysis tubing or Slide-A-Lyzer dialysis cassettes, 10K MWCO (ThermoFisher Scientific, MA) Gel-filtration columns: HiPrep 16/60 Sephacryl S-100 and Superdex 75 10/300 (GE HealthCare, NJ) Protein standards (Bio-Rad, CA) Anti-His antibody (Life Technologies, NY) Anti-tyrosinase antibody (T311 Santa Cruz Biotechnology, CA) Centrifuge 5415R (Eppendorf, Germany) UN-SCAN-IT gel ™ gel analysis software (Silk Scientific, Inc., UT) Stirring devices Centrifuge tubes Additional materials and equipment for SDS-PAGE ( UNIT 10.1 ) and Western blot ( UNIT 10.8 ) analysis. .. The DNA of hTyrCtr included residues 19-469 of the human tyrosinase, a TEV-protease cleavage site (NLYFQG sequence), and a 6×His-Tag ( ) was commercially synthesized, expressed in baculovirus and produced in whole insect T. ni larvae (Allotropic Tech., MD).

    Purification:

    Article Title: Characterization and Functional Analysis of atl, a Novel Gene Encoding Autolysin in Streptococcus suis
    Article Snippet: .. The supernatant was saved for subsequent purification of the 6×His-tagged fusion proteins using a HisTrap FF column (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) according to the manufacturer's instructions. ..

    Article Title: Bacterial expression of human kynurenine 3-monooxygenase: Solubility, activity, purification
    Article Snippet: .. The soluble supernatant fraction was purified on a 5 ml HisTrap ff crude column (GE Healthcare, 17-5286-01) using the AKTA protein purification system (GE Healthcare). .. The column was washed with ten column volumes of 20 mM HEPES pH7, 200 mM NaCl and 25 mM imidazole (Sigma Aldrich, I5513).

    Article Title: Characterization and Functional Analysis of atl, a Novel Gene Encoding Autolysin in Streptococcus suis
    Article Snippet: .. The Atl fusion protein was extracted from a mid-log culture, which had been induced with 1 mM IPTG for 5 h. The IPTG-inducible expression plasmid pET32a contained a start codon followed by a 6×His tag sequence, allowing purification of the recombinant autolysin by using a HisTrap FF column. .. As shown in , an apparent protein band of approximately 96 kDa appeared in the induced recombinant E. coli , while it was absent in the uninduced recombinant E. coli or induced E. coli containing control plasmid.

    Article Title: Large-scale seroprevalence analysis of human metapneumovirus and human respiratory syncytial virus infections in Beijing, China
    Article Snippet: .. The supernatant was purified using a HisTrap Q FF 5 ml column (GE Healthcare) followed by a HisTrap HP 1 ml column (GE Healthcare). .. The purification of N proteins was evaluated by 12% SDS-PAGE and optical density scanning using the Gene Genius Bio-imaging System (Syngene, Synoptics Ltd. Cambridge.

    Protein Purification:

    Article Title: Bacterial expression of human kynurenine 3-monooxygenase: Solubility, activity, purification
    Article Snippet: .. The soluble supernatant fraction was purified on a 5 ml HisTrap ff crude column (GE Healthcare, 17-5286-01) using the AKTA protein purification system (GE Healthcare). .. The column was washed with ten column volumes of 20 mM HEPES pH7, 200 mM NaCl and 25 mM imidazole (Sigma Aldrich, I5513).

    Fast Protein Liquid Chromatography:

    Article Title: Engineering of monobody conjugates for human EphA2-specific optical imaging
    Article Snippet: .. Supernatants were obtained by centrifugation at 10,000 rpm at 4°C and applied to a HisTrap FF column (GE Healthcare Biosciences, PA) in an AKTA FPLC system (GE Healthcare Biosciences). ..

    Expressing:

    Article Title: Characterization and Functional Analysis of atl, a Novel Gene Encoding Autolysin in Streptococcus suis
    Article Snippet: .. The Atl fusion protein was extracted from a mid-log culture, which had been induced with 1 mM IPTG for 5 h. The IPTG-inducible expression plasmid pET32a contained a start codon followed by a 6×His tag sequence, allowing purification of the recombinant autolysin by using a HisTrap FF column. .. As shown in , an apparent protein band of approximately 96 kDa appeared in the induced recombinant E. coli , while it was absent in the uninduced recombinant E. coli or induced E. coli containing control plasmid.

    Lysis:

    Article Title: The purification of recombinant human tyrosinase from insect larvae infected with the baculovirus vector
    Article Snippet: .. T.ni larvae biomass containing the protein of interest (Allotropic Tech., MD) Wet ice 50 ml tubes (Corning, NY) Lysis buffer (see recipe) 1-Phenyl-2-thiourea (PTU; Sigma-Aldrich, MO) DNase I (Thermo Fisher Scientific, PA) Complete set of protease inhibitors (Roche, USA) tris(2-carboxyethyl)phosphine (TCEP; Thermo Scientific, DE) Fisher Science Education pH meter (Thermo Fisher Scientific, MA) Omni Tissue Homogenizer TH (Omni International, GA) Hard Tissue Omni Tip™ Homogenizer Probes (Omni International, GA) Orbitron Rotator (Boekel Scientific, PA) Ultrasonic Processor GE130PB (Hielscher System, Germany) Sorvall Lynx 4000 Centrifuge (Thermo Scientific, DE) Rotor for centrifuge: Fiberlite, F21-8×50y (capacity 8 × 50 ml) Binding buffer (see recipe) Elution buffer (see recipe) Immobilized metal affinity (IMAC) column - HisTrap FF Crude (GE Healthcare, NJ) BioLogic Duo-Flow chromatography system (Bio-Rad, CA) Gel-filtration buffer (see recipe) NanoDrop 2000c UV-Vis spectrophotometer (Thermo Scientific, DE) 4-15% polyacrylamide gels (Bio-Rad, CA) 3,4-Dihydroxy-L-phenylalanine, L-DOPA (Sigma Aldrich, MO) MO) SpectraMax i3 Multi-Mode Microplate Detection Platform (Molecular Devices, CA) Clear-bottom polystyrene 96-well plates (Corning, NY) Dialysis membranes: SnakeSkin dialysis tubing or Slide-A-Lyzer dialysis cassettes, 10K MWCO (ThermoFisher Scientific, MA) Gel-filtration columns: HiPrep 16/60 Sephacryl S-100 and Superdex 75 10/300 (GE HealthCare, NJ) Protein standards (Bio-Rad, CA) Anti-His antibody (Life Technologies, NY) Anti-tyrosinase antibody (T311 Santa Cruz Biotechnology, CA) Centrifuge 5415R (Eppendorf, Germany) UN-SCAN-IT gel ™ gel analysis software (Silk Scientific, Inc., UT) Stirring devices Centrifuge tubes Additional materials and equipment for SDS-PAGE ( UNIT 10.1 ) and Western blot ( UNIT 10.8 ) analysis. .. The DNA of hTyrCtr included residues 19-469 of the human tyrosinase, a TEV-protease cleavage site (NLYFQG sequence), and a 6×His-Tag ( ) was commercially synthesized, expressed in baculovirus and produced in whole insect T. ni larvae (Allotropic Tech., MD).

    Sequencing:

    Article Title: Characterization and Functional Analysis of atl, a Novel Gene Encoding Autolysin in Streptococcus suis
    Article Snippet: .. The Atl fusion protein was extracted from a mid-log culture, which had been induced with 1 mM IPTG for 5 h. The IPTG-inducible expression plasmid pET32a contained a start codon followed by a 6×His tag sequence, allowing purification of the recombinant autolysin by using a HisTrap FF column. .. As shown in , an apparent protein band of approximately 96 kDa appeared in the induced recombinant E. coli , while it was absent in the uninduced recombinant E. coli or induced E. coli containing control plasmid.

    Western Blot:

    Article Title: The purification of recombinant human tyrosinase from insect larvae infected with the baculovirus vector
    Article Snippet: .. T.ni larvae biomass containing the protein of interest (Allotropic Tech., MD) Wet ice 50 ml tubes (Corning, NY) Lysis buffer (see recipe) 1-Phenyl-2-thiourea (PTU; Sigma-Aldrich, MO) DNase I (Thermo Fisher Scientific, PA) Complete set of protease inhibitors (Roche, USA) tris(2-carboxyethyl)phosphine (TCEP; Thermo Scientific, DE) Fisher Science Education pH meter (Thermo Fisher Scientific, MA) Omni Tissue Homogenizer TH (Omni International, GA) Hard Tissue Omni Tip™ Homogenizer Probes (Omni International, GA) Orbitron Rotator (Boekel Scientific, PA) Ultrasonic Processor GE130PB (Hielscher System, Germany) Sorvall Lynx 4000 Centrifuge (Thermo Scientific, DE) Rotor for centrifuge: Fiberlite, F21-8×50y (capacity 8 × 50 ml) Binding buffer (see recipe) Elution buffer (see recipe) Immobilized metal affinity (IMAC) column - HisTrap FF Crude (GE Healthcare, NJ) BioLogic Duo-Flow chromatography system (Bio-Rad, CA) Gel-filtration buffer (see recipe) NanoDrop 2000c UV-Vis spectrophotometer (Thermo Scientific, DE) 4-15% polyacrylamide gels (Bio-Rad, CA) 3,4-Dihydroxy-L-phenylalanine, L-DOPA (Sigma Aldrich, MO) MO) SpectraMax i3 Multi-Mode Microplate Detection Platform (Molecular Devices, CA) Clear-bottom polystyrene 96-well plates (Corning, NY) Dialysis membranes: SnakeSkin dialysis tubing or Slide-A-Lyzer dialysis cassettes, 10K MWCO (ThermoFisher Scientific, MA) Gel-filtration columns: HiPrep 16/60 Sephacryl S-100 and Superdex 75 10/300 (GE HealthCare, NJ) Protein standards (Bio-Rad, CA) Anti-His antibody (Life Technologies, NY) Anti-tyrosinase antibody (T311 Santa Cruz Biotechnology, CA) Centrifuge 5415R (Eppendorf, Germany) UN-SCAN-IT gel ™ gel analysis software (Silk Scientific, Inc., UT) Stirring devices Centrifuge tubes Additional materials and equipment for SDS-PAGE ( UNIT 10.1 ) and Western blot ( UNIT 10.8 ) analysis. .. The DNA of hTyrCtr included residues 19-469 of the human tyrosinase, a TEV-protease cleavage site (NLYFQG sequence), and a 6×His-Tag ( ) was commercially synthesized, expressed in baculovirus and produced in whole insect T. ni larvae (Allotropic Tech., MD).

    Binding Assay:

    Article Title: The purification of recombinant human tyrosinase from insect larvae infected with the baculovirus vector
    Article Snippet: .. T.ni larvae biomass containing the protein of interest (Allotropic Tech., MD) Wet ice 50 ml tubes (Corning, NY) Lysis buffer (see recipe) 1-Phenyl-2-thiourea (PTU; Sigma-Aldrich, MO) DNase I (Thermo Fisher Scientific, PA) Complete set of protease inhibitors (Roche, USA) tris(2-carboxyethyl)phosphine (TCEP; Thermo Scientific, DE) Fisher Science Education pH meter (Thermo Fisher Scientific, MA) Omni Tissue Homogenizer TH (Omni International, GA) Hard Tissue Omni Tip™ Homogenizer Probes (Omni International, GA) Orbitron Rotator (Boekel Scientific, PA) Ultrasonic Processor GE130PB (Hielscher System, Germany) Sorvall Lynx 4000 Centrifuge (Thermo Scientific, DE) Rotor for centrifuge: Fiberlite, F21-8×50y (capacity 8 × 50 ml) Binding buffer (see recipe) Elution buffer (see recipe) Immobilized metal affinity (IMAC) column - HisTrap FF Crude (GE Healthcare, NJ) BioLogic Duo-Flow chromatography system (Bio-Rad, CA) Gel-filtration buffer (see recipe) NanoDrop 2000c UV-Vis spectrophotometer (Thermo Scientific, DE) 4-15% polyacrylamide gels (Bio-Rad, CA) 3,4-Dihydroxy-L-phenylalanine, L-DOPA (Sigma Aldrich, MO) MO) SpectraMax i3 Multi-Mode Microplate Detection Platform (Molecular Devices, CA) Clear-bottom polystyrene 96-well plates (Corning, NY) Dialysis membranes: SnakeSkin dialysis tubing or Slide-A-Lyzer dialysis cassettes, 10K MWCO (ThermoFisher Scientific, MA) Gel-filtration columns: HiPrep 16/60 Sephacryl S-100 and Superdex 75 10/300 (GE HealthCare, NJ) Protein standards (Bio-Rad, CA) Anti-His antibody (Life Technologies, NY) Anti-tyrosinase antibody (T311 Santa Cruz Biotechnology, CA) Centrifuge 5415R (Eppendorf, Germany) UN-SCAN-IT gel ™ gel analysis software (Silk Scientific, Inc., UT) Stirring devices Centrifuge tubes Additional materials and equipment for SDS-PAGE ( UNIT 10.1 ) and Western blot ( UNIT 10.8 ) analysis. .. The DNA of hTyrCtr included residues 19-469 of the human tyrosinase, a TEV-protease cleavage site (NLYFQG sequence), and a 6×His-Tag ( ) was commercially synthesized, expressed in baculovirus and produced in whole insect T. ni larvae (Allotropic Tech., MD).

    SDS Page:

    Article Title: The purification of recombinant human tyrosinase from insect larvae infected with the baculovirus vector
    Article Snippet: .. T.ni larvae biomass containing the protein of interest (Allotropic Tech., MD) Wet ice 50 ml tubes (Corning, NY) Lysis buffer (see recipe) 1-Phenyl-2-thiourea (PTU; Sigma-Aldrich, MO) DNase I (Thermo Fisher Scientific, PA) Complete set of protease inhibitors (Roche, USA) tris(2-carboxyethyl)phosphine (TCEP; Thermo Scientific, DE) Fisher Science Education pH meter (Thermo Fisher Scientific, MA) Omni Tissue Homogenizer TH (Omni International, GA) Hard Tissue Omni Tip™ Homogenizer Probes (Omni International, GA) Orbitron Rotator (Boekel Scientific, PA) Ultrasonic Processor GE130PB (Hielscher System, Germany) Sorvall Lynx 4000 Centrifuge (Thermo Scientific, DE) Rotor for centrifuge: Fiberlite, F21-8×50y (capacity 8 × 50 ml) Binding buffer (see recipe) Elution buffer (see recipe) Immobilized metal affinity (IMAC) column - HisTrap FF Crude (GE Healthcare, NJ) BioLogic Duo-Flow chromatography system (Bio-Rad, CA) Gel-filtration buffer (see recipe) NanoDrop 2000c UV-Vis spectrophotometer (Thermo Scientific, DE) 4-15% polyacrylamide gels (Bio-Rad, CA) 3,4-Dihydroxy-L-phenylalanine, L-DOPA (Sigma Aldrich, MO) MO) SpectraMax i3 Multi-Mode Microplate Detection Platform (Molecular Devices, CA) Clear-bottom polystyrene 96-well plates (Corning, NY) Dialysis membranes: SnakeSkin dialysis tubing or Slide-A-Lyzer dialysis cassettes, 10K MWCO (ThermoFisher Scientific, MA) Gel-filtration columns: HiPrep 16/60 Sephacryl S-100 and Superdex 75 10/300 (GE HealthCare, NJ) Protein standards (Bio-Rad, CA) Anti-His antibody (Life Technologies, NY) Anti-tyrosinase antibody (T311 Santa Cruz Biotechnology, CA) Centrifuge 5415R (Eppendorf, Germany) UN-SCAN-IT gel ™ gel analysis software (Silk Scientific, Inc., UT) Stirring devices Centrifuge tubes Additional materials and equipment for SDS-PAGE ( UNIT 10.1 ) and Western blot ( UNIT 10.8 ) analysis. .. The DNA of hTyrCtr included residues 19-469 of the human tyrosinase, a TEV-protease cleavage site (NLYFQG sequence), and a 6×His-Tag ( ) was commercially synthesized, expressed in baculovirus and produced in whole insect T. ni larvae (Allotropic Tech., MD).

    Plasmid Preparation:

    Article Title: Characterization and Functional Analysis of atl, a Novel Gene Encoding Autolysin in Streptococcus suis
    Article Snippet: .. The Atl fusion protein was extracted from a mid-log culture, which had been induced with 1 mM IPTG for 5 h. The IPTG-inducible expression plasmid pET32a contained a start codon followed by a 6×His tag sequence, allowing purification of the recombinant autolysin by using a HisTrap FF column. .. As shown in , an apparent protein band of approximately 96 kDa appeared in the induced recombinant E. coli , while it was absent in the uninduced recombinant E. coli or induced E. coli containing control plasmid.

    Software:

    Article Title: The purification of recombinant human tyrosinase from insect larvae infected with the baculovirus vector
    Article Snippet: .. T.ni larvae biomass containing the protein of interest (Allotropic Tech., MD) Wet ice 50 ml tubes (Corning, NY) Lysis buffer (see recipe) 1-Phenyl-2-thiourea (PTU; Sigma-Aldrich, MO) DNase I (Thermo Fisher Scientific, PA) Complete set of protease inhibitors (Roche, USA) tris(2-carboxyethyl)phosphine (TCEP; Thermo Scientific, DE) Fisher Science Education pH meter (Thermo Fisher Scientific, MA) Omni Tissue Homogenizer TH (Omni International, GA) Hard Tissue Omni Tip™ Homogenizer Probes (Omni International, GA) Orbitron Rotator (Boekel Scientific, PA) Ultrasonic Processor GE130PB (Hielscher System, Germany) Sorvall Lynx 4000 Centrifuge (Thermo Scientific, DE) Rotor for centrifuge: Fiberlite, F21-8×50y (capacity 8 × 50 ml) Binding buffer (see recipe) Elution buffer (see recipe) Immobilized metal affinity (IMAC) column - HisTrap FF Crude (GE Healthcare, NJ) BioLogic Duo-Flow chromatography system (Bio-Rad, CA) Gel-filtration buffer (see recipe) NanoDrop 2000c UV-Vis spectrophotometer (Thermo Scientific, DE) 4-15% polyacrylamide gels (Bio-Rad, CA) 3,4-Dihydroxy-L-phenylalanine, L-DOPA (Sigma Aldrich, MO) MO) SpectraMax i3 Multi-Mode Microplate Detection Platform (Molecular Devices, CA) Clear-bottom polystyrene 96-well plates (Corning, NY) Dialysis membranes: SnakeSkin dialysis tubing or Slide-A-Lyzer dialysis cassettes, 10K MWCO (ThermoFisher Scientific, MA) Gel-filtration columns: HiPrep 16/60 Sephacryl S-100 and Superdex 75 10/300 (GE HealthCare, NJ) Protein standards (Bio-Rad, CA) Anti-His antibody (Life Technologies, NY) Anti-tyrosinase antibody (T311 Santa Cruz Biotechnology, CA) Centrifuge 5415R (Eppendorf, Germany) UN-SCAN-IT gel ™ gel analysis software (Silk Scientific, Inc., UT) Stirring devices Centrifuge tubes Additional materials and equipment for SDS-PAGE ( UNIT 10.1 ) and Western blot ( UNIT 10.8 ) analysis. .. The DNA of hTyrCtr included residues 19-469 of the human tyrosinase, a TEV-protease cleavage site (NLYFQG sequence), and a 6×His-Tag ( ) was commercially synthesized, expressed in baculovirus and produced in whole insect T. ni larvae (Allotropic Tech., MD).

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  • 99
    GE Healthcare histrap hp column
    Overexpression and purification of RbsB-His 6 and mutants DT002-His 6 and DT016-His 6 . ( A ) SDS-PAGE gel of purification steps with a <t>HisTrap</t> column of RbsB-His 6 . ( B ) and ( C ) as for panel ( A ) but for mutants DT0016-His 6 and DT002-His 6 , respectively. ( D ) SDS-PAGE gel of elution steps after adding 10 mM ATP and after gel filtration column for mutant DT016-His 6 . M, Marker; Elu, Elution step. Black triangle indicates the expected position of RbsB-His 6 , DT002-His 6 and DT016-His 6 proteins. Red triangle indicates the position of the assumed E. coli chaperones. Images in panels ( A – D ) stem from single individual SDS-PAGE gels, as indicated by the white line separator and panel lettering. Individual panel images and lanes were not further combined digitally and show the full protein size range.
    Histrap Hp Column, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 2134 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/histrap hp column/product/GE Healthcare
    Average 99 stars, based on 2134 article reviews
    Price from $9.99 to $1999.99
    histrap hp column - by Bioz Stars, 2021-01
    99/100 stars
      Buy from Supplier

    99
    GE Healthcare crude histrap column
    PKA phosphorylates GPKOW at S27 and T316 in vitro . A . Recombinant GPKOW proteins were expressed in bacteria and purified on a <t>HisTrap</t> column followed by an ion exchange column. GPKOW wt (lane 1), GPKOW S27A (lane 2), GPKOW T316A (lane 3) and GPKOW S27A+T316A (lane 4) were all recognized by anti-GPKOW B01 after separation on SDS-PAGE and immunoblotting. One representative immunoblot is shown. B . Purified GPKOW wt (lanes 1 and 2), single- (lanes 3-6) or double-mutated GPKOW (lanes 7 and 8) were incubated with active (+) or heat inactivated (-) PKA Cα1 (7.4 ng) and γ- 32 P-ATP in a reaction buffer. The samples were analyzed by SDS-PAGE and autoradiography. The CBB staining in the lower panel shows the amount of the different proteins. C . Unsaturated images from Syngene G-box and Typhoon 9400 phosphoimager were quantified by Genetools (Syngene) and the statistical analysis was performed with paired students T-test in GraphPad Prism. The asterisk indicates a significant difference between the columns with a p-value
    Crude Histrap Column, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/crude histrap column/product/GE Healthcare
    Average 99 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    crude histrap column - by Bioz Stars, 2021-01
    99/100 stars
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    Overexpression and purification of RbsB-His 6 and mutants DT002-His 6 and DT016-His 6 . ( A ) SDS-PAGE gel of purification steps with a HisTrap column of RbsB-His 6 . ( B ) and ( C ) as for panel ( A ) but for mutants DT0016-His 6 and DT002-His 6 , respectively. ( D ) SDS-PAGE gel of elution steps after adding 10 mM ATP and after gel filtration column for mutant DT016-His 6 . M, Marker; Elu, Elution step. Black triangle indicates the expected position of RbsB-His 6 , DT002-His 6 and DT016-His 6 proteins. Red triangle indicates the position of the assumed E. coli chaperones. Images in panels ( A – D ) stem from single individual SDS-PAGE gels, as indicated by the white line separator and panel lettering. Individual panel images and lanes were not further combined digitally and show the full protein size range.

    Journal: Scientific Reports

    Article Title: Computational redesign of the Escherichia coli ribose-binding protein ligand binding pocket for 1,3-cyclohexanediol and cyclohexanol

    doi: 10.1038/s41598-019-53507-5

    Figure Lengend Snippet: Overexpression and purification of RbsB-His 6 and mutants DT002-His 6 and DT016-His 6 . ( A ) SDS-PAGE gel of purification steps with a HisTrap column of RbsB-His 6 . ( B ) and ( C ) as for panel ( A ) but for mutants DT0016-His 6 and DT002-His 6 , respectively. ( D ) SDS-PAGE gel of elution steps after adding 10 mM ATP and after gel filtration column for mutant DT016-His 6 . M, Marker; Elu, Elution step. Black triangle indicates the expected position of RbsB-His 6 , DT002-His 6 and DT016-His 6 proteins. Red triangle indicates the position of the assumed E. coli chaperones. Images in panels ( A – D ) stem from single individual SDS-PAGE gels, as indicated by the white line separator and panel lettering. Individual panel images and lanes were not further combined digitally and show the full protein size range.

    Article Snippet: The clean lysate was next loaded onto a HisTrap HP column (HisTrap FF crude 1 ml, GE Healthcare) at 4 °C and flow rate of 0.5 ml min−1 , followed by washes of, consecutively, 10 column volumes (cv, equal to 1 ml) of buffer A with 20 mM imidazole, 1.5 cv of buffer A with 40 mM imidazole and 1.5 cv of buffer A with 80 mM imidazole.

    Techniques: Over Expression, Purification, SDS Page, Gel Purification, Filtration, Mutagenesis, Marker

    Chromatography profiles and enzymatic activities for the recombinant hTyrC tr and temperature sensitive mutant variant, R422Q A: IMAC using HisTrap FF crude 5 ml column with the linear gradient of 500 mM imidazole (gray line). B: Gel-filtration chromatography using HiPrep 16/60 Sephacryl S-100 column. The insert shows a magnification of A 280 profiles of hTyrC tr (black line) and R422Q mutant (green line). C: Gel filtration using Superdex 75 10/30 column. Top panels of A, B, and C show test tubes containing the L-DOPA colorimetric reactions for each protein fraction of hTyrC tr (black frame) and R422Q (green frame). Brown color (intensity proportional) in tube indicates diphenol oxidase activity.

    Journal: Current protocols in protein science

    Article Title: The purification of recombinant human tyrosinase from insect larvae infected with the baculovirus vector

    doi: 10.1002/cpps.37

    Figure Lengend Snippet: Chromatography profiles and enzymatic activities for the recombinant hTyrC tr and temperature sensitive mutant variant, R422Q A: IMAC using HisTrap FF crude 5 ml column with the linear gradient of 500 mM imidazole (gray line). B: Gel-filtration chromatography using HiPrep 16/60 Sephacryl S-100 column. The insert shows a magnification of A 280 profiles of hTyrC tr (black line) and R422Q mutant (green line). C: Gel filtration using Superdex 75 10/30 column. Top panels of A, B, and C show test tubes containing the L-DOPA colorimetric reactions for each protein fraction of hTyrC tr (black frame) and R422Q (green frame). Brown color (intensity proportional) in tube indicates diphenol oxidase activity.

    Article Snippet: T.ni larvae biomass containing the protein of interest (Allotropic Tech., MD) Wet ice 50 ml tubes (Corning, NY) Lysis buffer (see recipe) 1-Phenyl-2-thiourea (PTU; Sigma-Aldrich, MO) DNase I (Thermo Fisher Scientific, PA) Complete set of protease inhibitors (Roche, USA) tris(2-carboxyethyl)phosphine (TCEP; Thermo Scientific, DE) Fisher Science Education pH meter (Thermo Fisher Scientific, MA) Omni Tissue Homogenizer TH (Omni International, GA) Hard Tissue Omni Tip™ Homogenizer Probes (Omni International, GA) Orbitron Rotator (Boekel Scientific, PA) Ultrasonic Processor GE130PB (Hielscher System, Germany) Sorvall Lynx 4000 Centrifuge (Thermo Scientific, DE) Rotor for centrifuge: Fiberlite, F21-8×50y (capacity 8 × 50 ml) Binding buffer (see recipe) Elution buffer (see recipe) Immobilized metal affinity (IMAC) column - HisTrap FF Crude (GE Healthcare, NJ) BioLogic Duo-Flow chromatography system (Bio-Rad, CA) Gel-filtration buffer (see recipe) NanoDrop 2000c UV-Vis spectrophotometer (Thermo Scientific, DE) 4-15% polyacrylamide gels (Bio-Rad, CA) 3,4-Dihydroxy-L-phenylalanine, L-DOPA (Sigma Aldrich, MO) MO) SpectraMax i3 Multi-Mode Microplate Detection Platform (Molecular Devices, CA) Clear-bottom polystyrene 96-well plates (Corning, NY) Dialysis membranes: SnakeSkin dialysis tubing or Slide-A-Lyzer dialysis cassettes, 10K MWCO (ThermoFisher Scientific, MA) Gel-filtration columns: HiPrep 16/60 Sephacryl S-100 and Superdex 75 10/300 (GE HealthCare, NJ) Protein standards (Bio-Rad, CA) Anti-His antibody (Life Technologies, NY) Anti-tyrosinase antibody (T311 Santa Cruz Biotechnology, CA) Centrifuge 5415R (Eppendorf, Germany) UN-SCAN-IT gel ™ gel analysis software (Silk Scientific, Inc., UT) Stirring devices Centrifuge tubes Additional materials and equipment for SDS-PAGE ( UNIT 10.1 ) and Western blot ( UNIT 10.8 ) analysis.

    Techniques: Chromatography, Recombinant, Mutagenesis, Variant Assay, Filtration, Activity Assay

    Purified trKMO. (A) Chromatogram produced during NiNTA affinity purification of truncate KMO. (B) SDS-PAGE protein gel stained with simplyblue showing HisTrap purification of truncated human KMO protein, (a) Cell-free extract prior to affinity chromatography, (b) flowthrough proteins which did not bind to the column, (c) first column wash, (d) second column wash, (e) third column wash, (f) pure truncated human KMO protein (44 kDa) from the fractions under the chromatogram. (C) Cartoon illustrating the trKMO construct. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

    Journal: Protein Expression and Purification

    Article Title: Bacterial expression of human kynurenine 3-monooxygenase: Solubility, activity, purification

    doi: 10.1016/j.pep.2013.11.015

    Figure Lengend Snippet: Purified trKMO. (A) Chromatogram produced during NiNTA affinity purification of truncate KMO. (B) SDS-PAGE protein gel stained with simplyblue showing HisTrap purification of truncated human KMO protein, (a) Cell-free extract prior to affinity chromatography, (b) flowthrough proteins which did not bind to the column, (c) first column wash, (d) second column wash, (e) third column wash, (f) pure truncated human KMO protein (44 kDa) from the fractions under the chromatogram. (C) Cartoon illustrating the trKMO construct. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

    Article Snippet: The soluble supernatant fraction was purified on a 5 ml HisTrap ff crude column (GE Healthcare, 17-5286-01) using the AKTA protein purification system (GE Healthcare).

    Techniques: Purification, Produced, Affinity Purification, SDS Page, Staining, Affinity Chromatography, Construct

    PKA phosphorylates GPKOW at S27 and T316 in vitro . A . Recombinant GPKOW proteins were expressed in bacteria and purified on a HisTrap column followed by an ion exchange column. GPKOW wt (lane 1), GPKOW S27A (lane 2), GPKOW T316A (lane 3) and GPKOW S27A+T316A (lane 4) were all recognized by anti-GPKOW B01 after separation on SDS-PAGE and immunoblotting. One representative immunoblot is shown. B . Purified GPKOW wt (lanes 1 and 2), single- (lanes 3-6) or double-mutated GPKOW (lanes 7 and 8) were incubated with active (+) or heat inactivated (-) PKA Cα1 (7.4 ng) and γ- 32 P-ATP in a reaction buffer. The samples were analyzed by SDS-PAGE and autoradiography. The CBB staining in the lower panel shows the amount of the different proteins. C . Unsaturated images from Syngene G-box and Typhoon 9400 phosphoimager were quantified by Genetools (Syngene) and the statistical analysis was performed with paired students T-test in GraphPad Prism. The asterisk indicates a significant difference between the columns with a p-value

    Journal: Journal of Molecular Signaling

    Article Title: G-patch domain and KOW motifs-containing protein, GPKOW; a nuclear RNA-binding protein regulated by protein kinase A

    doi: 10.1186/1750-2187-6-10

    Figure Lengend Snippet: PKA phosphorylates GPKOW at S27 and T316 in vitro . A . Recombinant GPKOW proteins were expressed in bacteria and purified on a HisTrap column followed by an ion exchange column. GPKOW wt (lane 1), GPKOW S27A (lane 2), GPKOW T316A (lane 3) and GPKOW S27A+T316A (lane 4) were all recognized by anti-GPKOW B01 after separation on SDS-PAGE and immunoblotting. One representative immunoblot is shown. B . Purified GPKOW wt (lanes 1 and 2), single- (lanes 3-6) or double-mutated GPKOW (lanes 7 and 8) were incubated with active (+) or heat inactivated (-) PKA Cα1 (7.4 ng) and γ- 32 P-ATP in a reaction buffer. The samples were analyzed by SDS-PAGE and autoradiography. The CBB staining in the lower panel shows the amount of the different proteins. C . Unsaturated images from Syngene G-box and Typhoon 9400 phosphoimager were quantified by Genetools (Syngene) and the statistical analysis was performed with paired students T-test in GraphPad Prism. The asterisk indicates a significant difference between the columns with a p-value

    Article Snippet: The bacteria were lysed in Buffer A (50 mM sodium dihydrogen phosphate pH 7.5, 0.2 M NaCl, 5 mM Imidazole, 1 × EDTA free protease inhibitor cocktail (Roche 04693132001) and 0.4 mM betamercaptoetanol) and purified on a crude HisTrap column (GE Healthcare 11-0004-58) using Buffer A and Buffer B (50 mM sodium dihydrogen phosphate pH 7.5, 0.2 M NaCl, 500 mM Imidazole, 1 × EDTA free protease inhibitor cocktail (Roche 04693132001) and 0.4 mM betamercaptoetanol).

    Techniques: In Vitro, Recombinant, Purification, SDS Page, Incubation, Autoradiography, Staining