| Article ID | Journal | Published Year | Pages | File Type |
|---|---|---|---|---|
| 7557247 | Analytical Biochemistry | 2017 | 27 Pages |
Abstract
We demonstrate the utility of fluorescently labeled HC PhoS A197C for rapid-kinetics applications by measuring, using stopped-flow, the Pi release kinetics from LepA/EF4 following 70S ribosome-stimulated GTP hydrolysis. Altogether, the approach developed here allows for the high-yield and simplified in-house production of a Pi detection system suitable for rapid-kinetics approaches with comparable sensitivity to the commercially available Phosphate Sensor.
Keywords
phosMDCCNTPaseGTPaseBMEPSTPMSFIMACGTPNTPIPTGβ-mercaptoethanolEDTAEthylenediaminetetraacetic acidSDS-PAGESodium dodecyl sulfate polyacrylamide gel electrophoresisPhosphate releaseisopropyl β-D-1-thiogalactopyranosidenucleotide triphosphatePhosphate sensorinorganic phosphateFluorescencePhenylmethylsulfonylfluorideImmobilized metal-affinity chromatographyGuanosine triphosphateguanosine triphosphatase
Related Topics
Physical Sciences and Engineering
Chemistry
Analytical Chemistry
Authors
Dustin D. Smith, Dylan Girodat, Hans-Joachim Wieden, L. Brent Selinger,