Microcon 3000 molecular weight cut off ultrafilter

Example 1

NAD+ kinase served as the acceptor chemical and as a controller chemical. Adenosine triphosphate served as the donor chemical. Magnesium chloride and acetic acid served as controller chemicals. Phosphorylated NAD kinase served as the acceptor product. Adenosine diphosphate served as the donor product. A solution was prepared by combining 100 microliters of a solution of NAD kinase [880 micromolar] in water with a 200 microliters of a solution of adenosine triphosphate [5 millimolar], magnesium chloride [10 millimolar], tris(hydroxymethyl)aminomethane buffer [100 millimolar, pH 7.5] in water (FIG. 1, Box 1). The reaction was allowed to incubate for 10 seconds, after which 200 microliters of a solution of 5% acetic acid in water was added (FIG. 1, Box 2). The acceptor product was separated from the adenosine triphosphate and adenosine diphosphate using a Microcon 3000 molecular weight cut off (MWCO) ultrafilter, available from Millipore Corporate Headquarters, 290 Concord Road, Billerica, Mass. 01821, USA (FIG. 1, Box 3). The NADP was measured using an x-ray fluorescence spectrometer equipped with a 50 watt chromium anode x-ray tube and a silicon drift detector (FIG. 1, Box 4). The ratio of the phosphorus x-ray fluorescence signal to sulfur x-ray fluorescence signal was 0.030501 for the NAD, and 0.264657 for the phosphorylated NAD.

Example 1

NAD+ kinase served as the acceptor chemical and as a controller chemical. Adenosine triphosphate served as the donor chemical. Magnesium chloride and acetic acid served as controller chemicals. Phosphorylated NAD kinase served as the acceptor product. Adenosine diphosphate served as the donor product. A solution was prepared by combining 100 microliters of a solution of NAD kinase [880 micromolar] in water with a 200 microliters of a solution of adenosine triphosphate [5 millimolar], magnesium chloride [10 millimolar], tris(hydroxymethyl)aminomethane buffer [100 millimolar, pH 7.5] in water (FIG. 1, Box 1). The reaction was allowed to incubate for 10 seconds, after which 200 microliters of a solution of 5% acetic acid in water was added (FIG. 1, Box 2). The acceptor product was separated from the adenosine triphosphate and adenosine diphosphate using a Microcon 3000 molecular weight cut off (MWCO) ultrafilter, available from Millipore Corporate Headquarters, 290 Concord Road, Billerica, Mass. 01821, USA (FIG. 1, Box 3). The NADP was measured using an x-ray fluorescence spectrometer equipped with a 50 watt chromium anode x-ray tube and a silicon drift detector (FIG. 1, Box 4). The ratio of the phosphorus x-ray fluorescence signal to sulfur x-ray fluorescence signal was 0.030501 for the NAD, and 0.264657 for the phosphorylated NAD.