Erika Hawkins
Promega
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Featured researches published by Erika Hawkins.
ChemBioChem | 2008
Wenhui Zhou; Christine Ann Andrews; Jianquan Liu; John Shultz; Michael P. Valley; Jim Cali; Erika Hawkins; Dieter Klaubert; Robert F. Bulleit; Keith V. Wood
Alkaline phosphatase (AP)—a stable enzyme with high specific activity for the hydrolysis of phosphate esters—is widely used as a conjugated enzyme label in enzyme-linked immunosorbent assays (ELISA) and DNA hybridization assays. It is also used as an in situ probe to monitor the expression and translocation of fusion proteins from the cytoplasm and for visualization of the spatial distribution of target biomolecules, such as cognate ligands or receptors in cells, tissues, and embryos. Among the many methods for detecting AP activity, there are various phosphate substrates, such as the colorimetric p-nitrophenyl phosphate, the fluorescent AttoPhos<, and the chemiluminescent adamantyl 1,2-diACHTUNGTRENNUNGoxetane AMPPD derivatives (Scheme 1). It is the ultrasensitivity of chemiluminescence, specifically with 1,2-dioxetane AMPPD derivatives, that has made this the overwhelming choice for monitoring AP activity. Although a luciferase-coupled bioluminescent assay is not only generically similar to the chemiluminescent assay and could show similar sensitivity, it also has the additional potential of creating recombinant luciferase to AP protein fusions, which might be preferable for the detection of AP activity in situ. The development of a suitable substrate to reach this ultrasensitivity is needed in order to promote the bioluminescent AP assay for practical applications. Chemical modification of the 6-hydroxyl group of luciferin (or the 6-amino group of aminoluciferin) is an effective means to approach bioluminescent assays for enzymes of interest, and 6-luciferin phosphate (Scheme 1) has been previously shown to detect AP activity. However, the detection limit of 10 19 mol of AP was 2–3 orders of magnitude lower than that for the AMPPD assay. Since the hydrolysis of phosphate monoesters is highly dependent on the pKa of the leaving group and the lower pKa 8.5 [11] of the luciferin phenol compared to a pKa ~9.0 of the adamantyl dioxetane phenol favors both nucleophilic attack and P O bond fission Scheme 1. Chemical structures of substrates for AP enzyme. A) Known chemiluminescent substrate AMPPD derivatives and bioluminescent substrate 6-luciferin phosphate; B) proposed self-cleavable luciferin phosphates, aminoluciferin trimethyl lock phosphate 1, and luciferin p-hydroxymethylphenyl phosphate 2.
Journal of the American Chemical Society | 2006
Wenhui Zhou; Michael P. Valley; John Shultz; Erika Hawkins; Laurent Bernad; Troy Good; Dave Good; Terry Riss; Dieter Klaubert; Keith V. Wood
Analytical Biochemistry | 2006
Michael P. Valley; Wenhui Zhou; Erika Hawkins; John Shultz; James J. Cali; Tracy J Worzella; Laurent Bernad; Troy Good; Dave Good; Terry Riss; Dieter Klaubert; Keith V. Wood
Archive | 2006
James J. Cali; William J. Daily; Erika Hawkins; Dieter Klaubert; Jianquan Liu; Poncho Meisenheimer; Michael Scurria; John Shultz; James Unch; Michael P. Valley; Keith V. Wood; Wenhui Zhou
Archive | 2003
James J. Cali; Dieter Klaubert; William J. Daily; Samuel Kin Sang Ho; Susan Frackman; Erika Hawkins; Keith V. Wood
Chemical Communications | 2006
Wenhui Zhou; John Shultz; Nancy Murphy; Erika Hawkins; Laurent Bernad; Troy Good; Leonard Moothart; Susan Frackman; Dieter Klaubert; Robert F. Bulleit; Keith V. Wood
Archive | 2013
Erika Hawkins; James J. Cali; Samuel Kin Sang Ho; Martha O'Brien; Richard Somberg; Robert F. Bulleit; Keith V. Wood
Archive | 2004
Erika Hawkins; John M. Centanni; Jacqueline Sankbeil; Keith V. Wood
Archive | 2002
Keith V. Wood; Erika Hawkins; Mike Scurria; Dieter Klaubert
Archive | 2008
William J. Daily; Erika Hawkins; Dieter Klaubert; Mark McDougall; James Unch; Keith V. Wood; Wenhui Zhou; Ji Zhu