Journal: Journal of Lipids

Article Title: A Nonradioactive Fluorimetric SPE-Based Ceramide Kinase Assay Using NBD-C 6 -Ceramide

doi: 10.1155/2012/404513

Figure Lengend Snippet: Structure of C 6 -ceramide and NBD-C 6 -ceramide.

Article Snippet: After acidic phase separation, the lower phase was dried and the phosphorylated NBD-C 6 -Cer was further purified by preparative TLC (silica G60; Merck) in solvent B, followed by elution with chloroform/methanol/water (5/5/1, v/v).

Techniques:

Journal: Journal of Lipids

Article Title: A Nonradioactive Fluorimetric SPE-Based Ceramide Kinase Assay Using NBD-C 6 -Ceramide

doi: 10.1155/2012/404513

Figure Lengend Snippet: Influence of water and acidity on fluorescence of NBD-derivatives. Fluorescence of methanolic NBD-C 6 -Cer-1-P solutions (1 μ M, 200 μ L) containing increasing amounts of water (a) different acids at 0.5 N final concentration (b) or increasing TFA concentration (c) ( λ ex 465 nm, λ em 535 nm; Tecan Infinite 200).

Article Snippet: After acidic phase separation, the lower phase was dried and the phosphorylated NBD-C 6 -Cer was further purified by preparative TLC (silica G60; Merck) in solvent B, followed by elution with chloroform/methanol/water (5/5/1, v/v).

Techniques: Fluorescence, Concentration Assay

Journal: Journal of Lipids

Article Title: A Nonradioactive Fluorimetric SPE-Based Ceramide Kinase Assay Using NBD-C 6 -Ceramide

doi: 10.1155/2012/404513

Figure Lengend Snippet: Separation of NBD-C 6 -Cer and NBD-C 6 -Cer-1-P via NH 2 -SPE. A mixture containing NBD-C 6 -Cer and NBD-C 6 -Cer-1-P, both at 5 μ M (a) or at a total concentration of 5 μ M but with a variable ratio (b) in the assay medium was separated via NH 2 -SPE, as described in . The eluted fractions were dried, resolubilized in chloroform/methanol (1/2, v/v) and the lipids were separated on silica G TLC plates (chloroform/acetone/methanol/acetic acid/water, 10/4/3/2/1, v/v), followed by fluorescence scanning of the spots (NBD-C 6 -Cer (black bars); NBD-C 6 -Cer-1-P (grey bars).) The result is expressed as percentage of total fluorescence in the elution fraction (a) mean ± SEM; n = 5; (b) single experiment).

Article Snippet: After acidic phase separation, the lower phase was dried and the phosphorylated NBD-C 6 -Cer was further purified by preparative TLC (silica G60; Merck) in solvent B, followed by elution with chloroform/methanol/water (5/5/1, v/v).

Techniques: Concentration Assay, Fluorescence

Journal: Journal of Lipids

Article Title: A Nonradioactive Fluorimetric SPE-Based Ceramide Kinase Assay Using NBD-C 6 -Ceramide

doi: 10.1155/2012/404513

Figure Lengend Snippet: Substrate and time dependence of the kinase reaction. Recombinant bacterially expressed Hs CERK was incubated with the indicated concentration of NBD-C 6 -Cer (a) or 5 μ M (b) in the presence of 1 mM ATP at 37°C. The reaction was stopped at 10 min (a) or the indicated time periods (b) by addition of methanol and the mixture was applied to an NH 2 -SPE column. The reaction product NBD-C 6 -Cer-1-P was quantified based on fluorescence intensity of the column eluate and converted into nmol or μ M, based on a calibration curve.

Article Snippet: After acidic phase separation, the lower phase was dried and the phosphorylated NBD-C 6 -Cer was further purified by preparative TLC (silica G60; Merck) in solvent B, followed by elution with chloroform/methanol/water (5/5/1, v/v).

Techniques: Recombinant, Incubation, Concentration Assay, Fluorescence

Journal: Journal of Lipids

Article Title: A Nonradioactive Fluorimetric SPE-Based Ceramide Kinase Assay Using NBD-C 6 -Ceramide

doi: 10.1155/2012/404513

Figure Lengend Snippet: CERK activity in cultured cells. CERK activity towards NBD-C 6 -Cer was determined in lysates from CHO cells, transfected with pCMV-Tag2B (mock) or pHVO001, coding for a Flag-HsCERK fusion , using the fluorescence assay (black bars) or the radiometric assay (grey bars, mean ± SEM, n = 3). CERK activity is expressed as nmol per mg protein per min (nmol/min·mg protein).

Article Snippet: After acidic phase separation, the lower phase was dried and the phosphorylated NBD-C 6 -Cer was further purified by preparative TLC (silica G60; Merck) in solvent B, followed by elution with chloroform/methanol/water (5/5/1, v/v).

Techniques: Activity Assay, Cell Culture, Transfection, Fluorescence

Journal: Journal of Lipids

Article Title: A Nonradioactive Fluorimetric SPE-Based Ceramide Kinase Assay Using NBD-C 6 -Ceramide

doi: 10.1155/2012/404513

Figure Lengend Snippet: Influence of proteine kinase inhibitors on CERK via 96 well HTS. CERK was measured with NBD-C 6 -Cer in the presence of 0.5 mM of established protein kinase inhibitors (kinase inhibitor library, Biomol), using the 96 well fluorescent SPE assay. Activity is expressed as % of the control containing 10% DMSO (100 ± 11%; mean ± SD; n = 3, magenta bars). CERK was not influenced by DMSO, up to 20% (data not shown). For comparison, effect of the inhibitors on sphingosine kinase is displayed in blue bars as % of control (100 ± 7%; mean ± SD; n = 3).

Article Snippet: After acidic phase separation, the lower phase was dried and the phosphorylated NBD-C 6 -Cer was further purified by preparative TLC (silica G60; Merck) in solvent B, followed by elution with chloroform/methanol/water (5/5/1, v/v).

Techniques: Activity Assay