histrap ff crude column  (GE Healthcare)

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

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

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

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