n terminal his 6 affinity tag  (Twist Bioscience)

Journal: ChemMedChem

Article Title: Developing an irreversible inhibitor of human DDAH-1, an enzyme upregulated in melanoma

doi: 10.1002/cmdc.201300557

Figure Lengend Snippet: Substrate and inhibitors of DDAH-1. Shown are the substrate Nω,Nω-dimethyl-L-arginine (ADMA), the covalent reversible inhibitor N5-(1-iminopropyl)- L-ornithine (IPO), the covalent irreversible inhibitor chloroacetamidine (CAA), and the covalent irreversible inhibitor N5-(1-imino, 2-chloroethyl)-L-ornithine (Cl-NIO).

Article Snippet: Materials Unless specified otherwise, all chemicals were from the Sigma Aldrich Chemical Co. Wild-type human DDAH-1 bearing an N -terminal His 6 affinity tag was expressed using an expression plasmid with a reengineeried N -terminus (pET28a-hDDAH-1re) to avoid N -terminal nonenzymatic gluconoylation, and purified as described previously.

Techniques:

Journal: ChemMedChem

Article Title: Developing an irreversible inhibitor of human DDAH-1, an enzyme upregulated in melanoma

doi: 10.1002/cmdc.201300557

Figure Lengend Snippet: Inactivation of purified DDAH-1 by Cl-NIO. A) Time- and concentration-dependent inactivation of purified DDAH-1 is observed when assayed in the presence of competing substrate and 0 (○), 20 (□), 40 (◆), 80 (▼), 100 (+), 150 (△), 300 (●), and 600 (■) μM of Cl-NIO. Solid lines are fits as described in Experimental Methods. B) The inverse of the apparent inactivation rates (A) are replotted as described in Experimental Methods to derive KI (1.3 ± 0.6 μM) and kinact (0.34 ± 0.07 min−1) values for Cl-NIO inactivation of DDAH-1.

Article Snippet: Materials Unless specified otherwise, all chemicals were from the Sigma Aldrich Chemical Co. Wild-type human DDAH-1 bearing an N -terminal His 6 affinity tag was expressed using an expression plasmid with a reengineeried N -terminus (pET28a-hDDAH-1re) to avoid N -terminal nonenzymatic gluconoylation, and purified as described previously.

Techniques: Purification, Concentration Assay

Journal: ChemMedChem

Article Title: Developing an irreversible inhibitor of human DDAH-1, an enzyme upregulated in melanoma

doi: 10.1002/cmdc.201300557

Figure Lengend Snippet: Studies of Cl-NIO-treated HEK293T cells. A) Two-color Western blot (upper panel) shows fluorescence derived from response to a myc-tag genetically encoded into an episomally-expressed DDAH-1 (red), and from response to a biotin-tagged activity probe for DDAH-1 (green), when HEK239T cells are treated by increasing concentrations of Cl-NIO (left to right: 0, 5, 10, 20, 40, 80 μM). Normalized fluorescence values for the biotin-derived signal (bottom panel) are fit to give an apparent “in cell” IC50 value for DDAH-1 inhibition of 6.6 ± 0.2 μM and a Hill coefficient of 1.8 ± 0.1. B) Toxicity of Cl-NIO to the HEK293T cell line after a 72 h incubation was assessed using an MTS assay (see Materials) to determine an IC50 = 92 ± 2 μM for cell survival.

Article Snippet: Materials Unless specified otherwise, all chemicals were from the Sigma Aldrich Chemical Co. Wild-type human DDAH-1 bearing an N -terminal His 6 affinity tag was expressed using an expression plasmid with a reengineeried N -terminus (pET28a-hDDAH-1re) to avoid N -terminal nonenzymatic gluconoylation, and purified as described previously.

Techniques: Western Blot, Fluorescence, Derivative Assay, Activity Assay, Inhibition, Incubation, MTS Assay

Journal: ChemMedChem

Article Title: Developing an irreversible inhibitor of human DDAH-1, an enzyme upregulated in melanoma

doi: 10.1002/cmdc.201300557

Figure Lengend Snippet: Assays for DDAH expression in melanoma cell lines. Immunohistochemical staining for DDAH-1 is applied various cell lines to assay for blue color due to the hematoxylin counter stain or for a brown color arising from a primary DDAH-1 antibody / peroxidase-linked secondary antibody (see Methods): A) normal human epidermal melanocytes, B) A375. A table summarizing staining for DDAH-1, DDAH-2 and iNOS in > 60 cell lines is found in Supporting Information (Table S1). C) Western blot for DDAH-1, DDAH-2 and actin (as a control for equal loading) in selected cell lines.

Article Snippet: Materials Unless specified otherwise, all chemicals were from the Sigma Aldrich Chemical Co. Wild-type human DDAH-1 bearing an N -terminal His 6 affinity tag was expressed using an expression plasmid with a reengineeried N -terminus (pET28a-hDDAH-1re) to avoid N -terminal nonenzymatic gluconoylation, and purified as described previously.

Techniques: Expressing, Immunohistochemical staining, Staining, Western Blot

Journal: ChemMedChem

Article Title: Developing an irreversible inhibitor of human DDAH-1, an enzyme upregulated in melanoma

doi: 10.1002/cmdc.201300557

Figure Lengend Snippet: Treatment of A375 cells with Cl-NIO. A) Immunohistochemical staining of A375 cells for 3-nitrotyrosine, DDAH-1 and iNOS, with and without treatment by Cl-NIO. The hematoxylin counterstain (negative result) is blue and the positive result is brown, due to the activity of a peroxidase-linked secondary antibody bound to separate primary antibodies for each analyte. B) Total nitrate and nitrite were measured in A375 culture supernatants after treatment by various concentrations of Cl-NIO, as described in Methods.

Article Snippet: Materials Unless specified otherwise, all chemicals were from the Sigma Aldrich Chemical Co. Wild-type human DDAH-1 bearing an N -terminal His 6 affinity tag was expressed using an expression plasmid with a reengineeried N -terminus (pET28a-hDDAH-1re) to avoid N -terminal nonenzymatic gluconoylation, and purified as described previously.

Techniques: Immunohistochemical staining, Staining, Activity Assay