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Probing enzymatic activity inside single cells.

Jessica Olofsson ; Shijun Xu (Institutionen för kemi- och bioteknik, Fysikalisk kemi) ; Gavin Jeffries (Institutionen för kemi- och bioteknik, Fysikalisk kemi) ; Aldo Jesorka (Institutionen för kemi- och bioteknik, Fysikalisk kemi) ; Helen Bridle ; Ida Isaksson ; Stephen G Weber ; Owe Orwar (Institutionen för kemi- och bioteknik, Fysikalisk kemi)
Analytical chemistry (1520-6882). Vol. 85 (2013), 21, p. 10126-33.
[Artikel, refereegranskad vetenskaplig]

We report a novel approach for determining the enzymatic activity within a single suspended cell. Using a steady-state microfluidic delivery device and timed exposure to the pore-forming agent digitonin, we controlled the plasma membrane permeation of individual NG108-15 cells. Mildly permeabilized cells (∼100 pores) were exposed to a series of concentrations of fluorescein diphosphate (FDP), a fluorogenic alkaline phosphatase substrate, with and without levamisole, an alkaline phosphatase inhibitor. We generated quantitative estimates for intracellular enzyme activity and were able to construct both dose-response and dose-inhibition curves at the single-cell level, resulting in an apparent Michaelis contant Km of 15.3 μM ± 1.02 (mean ± standard error of the mean (SEM), n = 16) and an inhibition constant Ki of 0.59 mM ± 0.07 (mean ± SEM, n = 14). Enzymatic activity could be monitored just 40 s after permeabilization, and five point dose-inhibition curves could be obtained within 150 s. This rapid approach offers a new methodology for characterizing enzyme activity within single cells.



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Denna post skapades 2013-11-18. Senast ändrad 2016-06-30.
CPL Pubid: 186993

 

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Institutioner (Chalmers)

Institutionen för kemi- och bioteknik, Fysikalisk kemi (2005-2014)

Ämnesområden

Nanovetenskap och nanoteknik
Biofysikalisk kemi

Chalmers infrastruktur