The publication feed below is often incomplete and out of date; for an up to date summary of our publications please see Google Scholar or Pub Med

Citation

BibTex format

@article{Lanyon-Hogg:2015:10.1016/j.ab.2015.08.025,
author = {Lanyon-Hogg, T and Masumoto, N and Bodakh, G and Konitsiotis, AD and Thinon, E and Rodgers, UR and Owens, RJ and Magee, AI and Tate, EW},
doi = {10.1016/j.ab.2015.08.025},
journal = {Analytical Biochemistry},
pages = {66--72},
title = {Click chemistry armed enzyme-linked immunosorbent assay to measure palmitoylation by hedgehog acyltransferase},
url = {http://dx.doi.org/10.1016/j.ab.2015.08.025},
volume = {490},
year = {2015}
}
Download

RIS format (EndNote, RefMan)

TY  - JOUR
AB  - Hedgehog signaling is critical for correct embryogenesis and tissue development. However, on maturation, signaling is also found to be aberrantly activated in many cancers. Palmitoylation of the secreted signaling protein sonic hedgehog (Shh) by the enzyme hedgehog acyltransferase (Hhat) is required for functional signaling. To quantify this important posttranslational modification, many in vitro Shh palmitoylation assays employ radiolabeled fatty acids, which have limitations in terms of cost and safety. Here we present a click chemistry armed enzyme-linked immunosorbent assay (click–ELISA) for assessment of Hhat activity through acylation of biotinylated Shh peptide with an alkyne-tagged palmitoyl-CoA (coenzyme A) analogue. Click chemistry functionalization of the alkyne tag with azido-FLAG peptide allows analysis through an ELISA protocol and colorimetric readout. This assay format identified the detergent n-dodecyl β-d-maltopyranoside as an improved solubilizing agent for Hhat activity. Quantification of the potency of RU-SKI small molecule Hhat inhibitors by click–ELISA indicated IC50 values in the low- or sub-micromolar range. A stopped assay format was also employed that allows measurement of Hhat kinetic parameters where saturating substrate concentrations exceed the binding capacity of the streptavidin-coated plate. Therefore, click–ELISA represents a nonradioactive method for assessing protein palmitoylation in vitro that is readily expandable to other classes of protein lipidation.
AU  - Lanyon-Hogg,T
AU  - Masumoto,N
AU  - Bodakh,G
AU  - Konitsiotis,AD
AU  - Thinon,E
AU  - Rodgers,UR
AU  - Owens,RJ
AU  - Magee,AI
AU  - Tate,EW
DO  - 10.1016/j.ab.2015.08.025
EP  - 72
PY  - 2015///
SN  - 1096-0309
SP  - 66
TI  - Click chemistry armed enzyme-linked immunosorbent assay to measure palmitoylation by hedgehog acyltransferase
T2  - Analytical Biochemistry
UR  - http://dx.doi.org/10.1016/j.ab.2015.08.025
UR  - http://hdl.handle.net/10044/1/38428
VL  - 490
ER  -
Download

Contact

Prof. Ed Tate
GSK Chair in Chemical Biology
Department of Chemistry
Molecular Sciences Research Hub, White City Campus,
82 Wood Lane, London, W12 0BZ

e.tate@imperial.ac.uk
Tel: +44 (0)20 759 + ext 43752 or 45821