Journal: PLoS ONE

Article Title: Molecular Identification of Carnosine N-Methyltransferase as Chicken Histamine N-Methyltransferase-Like Protein (HNMT-Like)

doi: 10.1371/journal.pone.0064805

Figure Lengend Snippet: Chicken HNMT-like protein was produced in COS-7 cells and purified to homogeneity by affinity chromatography on nickel-sepharose (HisTrap HP) as described under “ ”. For the SDS-PAGE analysis (A), 20 µl of reduced sample from each fraction was loaded onto a 10% gel, electrophoresed and the resulting gel was then stained with silver . For the Western-blot analysis (B), 1 µl of reduced sample from each fraction was loaded onto a 10% gel, electrophoresed and blotted to nitrocellulose membrane which was then sequentially probed with a mouse primary antibody against His6 tag and a horseradish peroxidase-conjugated goat anti-mouse antibody. Secondary antibody was detected through autoradiography using chemiluminescence. M, prestained protein marker; L, cell-free lysate of COS-7 cells overexpressing the recombinant enzyme; AL, 4-fold diluted lysate applied to the column; FT, flow through; W, wash; Fractions 30 to 300 were eluted with the indicated concentrations of imidazole. The pattern of prestained protein bands was copied from the blotting membrane onto ECL film using a set of felt-tip pens.

Article Snippet: For transfections, COS-7 cells (CLS Cell Lines Service GmbH, Germany) or HEK-293T (a kind gift of Dr. Maria Veiga-da-Cunha, de Duve Institute, Brussels) were plated in 100-mm Petri dishes at a cell density of 1.7×10 6 or 2.1×10 6 cells per plate, respectively, in Dulbecco's minimal essential medium supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin, and 10% (v/v) fetal bovine serum, and grown in a humidified incubator under 95% air and 5% CO 2 atmosphere at 37°C.

Techniques: Produced, Purification, Affinity Chromatography, SDS Page, Staining, Western Blot, Membrane, Autoradiography, Marker, Recombinant

Journal: PLoS ONE

Article Title: Molecular Identification of Carnosine N-Methyltransferase as Chicken Histamine N-Methyltransferase-Like Protein (HNMT-Like)

doi: 10.1371/journal.pone.0064805

Figure Lengend Snippet: COS-7 and HEK-293T cells were transfected with either unmodified pEF6/Myc-His A vector (Control) or the same vector encoding chicken HNMT-like protein (HNMT-like) as described under “ ”. The cell-free lysates (2–3 µg of protein) were incubated for 0, 5, 10, 15 and 20 min in the reaction mixture containing 1 µM SAM (100 pmol), as well as 440×10 3 cpm of ( 3 H)SAM. The formation of radiolabeled anserine was determined after its chromatographic separation from ( 3 H)SAM. Values are the means ± S.E. of two separate transfection experiments. The presence of recombinant protein in tested lysates was verified by Western-blot analysis. Lysates (15 µg of protein) were loaded reduced onto a 10% gel, electrophoresed and blotted to nitrocellulose membrane which was then sequentially probed with a mouse primary antibody against His6 tag and a horseradish peroxidase-conjugated goat anti-mouse antibody. Secondary antibody was detected through autoradiography using chemiluminescence. COS, COS-7 cell lysate; HEK, HEK-293T cell lysate.

Article Snippet: For transfections, COS-7 cells (CLS Cell Lines Service GmbH, Germany) or HEK-293T (a kind gift of Dr. Maria Veiga-da-Cunha, de Duve Institute, Brussels) were plated in 100-mm Petri dishes at a cell density of 1.7×10 6 or 2.1×10 6 cells per plate, respectively, in Dulbecco's minimal essential medium supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin, and 10% (v/v) fetal bovine serum, and grown in a humidified incubator under 95% air and 5% CO 2 atmosphere at 37°C.

Techniques: Transfection, Plasmid Preparation, Control, Incubation, Recombinant, Western Blot, Membrane, Autoradiography