Alkaline phosphatase, single molecule assay

A control solution of 8xl0 M alkaline phosphatase was prepared. A second solution of 8xl0 ° M enzyme was heated at 64°C for 5 min and immediately diluted by 6 orders of magnitude. Both samples were assayed by the single molecule assay method. 

Figure 1 Single Molecule Assay (A) Blank generated by the 3 min injection at 400 Vcm of 1AttoPhos in 100 mM borate (pH 9.5). Following an incubation of 19.25 min, the sample was swept past the detector at 400Vcm . (B) 1.9x10 M alkaline phosphatase mixed with 1 mM AttoPhos in 100 mM borate (pH 9.5). Sample was treated as in (A) but with a 28.5 min incubation. (C) 9.5xl0 M alkal e phosphatase mixed with 1 mM AttoPhos in 100 mM borate (pH 9.5). Sample was treated as in (A) but with a 18 min incubation.

Partially denatured alkaline phosphatase was assayed and compared to control enzyme. With a 15 min incubation of 8x10 M control enzyme, 9.8 3.3 peaks were observed per assay (n=4) with activities of 138 107 s . This is about twice the number observed in the single molecule assays different enzyme lots were used for the two studies. Denatured enzyme produced 4.4 1.5 peaks/assay (n=4) with an average activity of 118 97 s . At the 85% confidence limit, the means of the activities are identical and at the 75% confidence limit the standard deviations are identical. A 50% loss of the activity in bulk assay results in 45 9%... 

SINGLE ALKALINE PHOSPHATASE MOLECULE ASSAY BY CAPILLARY ELECTROPHORESIS LASER-INDUCED FLUORESCENCE DETECTION... 

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