Abstract：
:S-palmitoylation (S-acylation) is emerging as an important dynamic post-translational modification of cysteine residues within proteins. Current assays for protein S-palmitoylation involve either in vivo labeling or chemical cleavage of S-palmitoyl groups to reveal a free cysteine sulfhydryl that can be subsequently labeled with an affinity handle (acyl-exchange). Assays for protein S-palmitoylation using acyl-exchange chemistry therefore require blocking of non-S-palmitoylated cysteines, typically using N-ethylmaleimide (NEM), to prevent non-specific detection. This in turn necessitates multiple precipitation-based clean-up steps to remove reagents between stages, often leading to variable sample loss, reduced signal, or protein aggregation. These combine to reduce the sensitivity, reliability, and accuracy of these assays, which also require a substantial amount of time to perform. By substituting these precipitation steps with chemical scavenging of NEM by 2,3-dimethyl-1,3-butadiene in an aqueous Diels-Alder 4+2 cyclo-addition reaction, it is possible to greatly improve sensitivity and accuracy while reducing the hands-on time and overall time required for the assay.
journal_name
Biotechniquesjournal_title
BioTechniquesauthors
Hurst CH,Turnbull D,Plain F,Fuller W,Hemsley PAdoi
10.2144/000114516subject
Has Abstractpub_date
2017-02-01 00:00:00pages
69-75issue
2eissn
0736-6205issn
1940-9818pii
000114516journal_volume
62pub_type
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