| Products/Services Used | Details | Operation |
|---|---|---|
| Catalog Antibody | To prevent the formation of disulfide bonds, 300 μM of FCM-ybbR (GenScript, USA) was incubated with 2 mM tris-2-carboxyethyl-phosphine (TCEP, Sigma Aldrich, cat. No. C4706) in a solution buffered at pH 7.0 (phosphate buffered saline, PBS, 0 M NaCl) and flushed with argon gas for 15 minutes. FlAsH-EDT2 was then added to a concentration of 27 μM, which resulted in 10x molar excess of FCM-ybbR (270 μM). This solution was gently stirred and incubated in the dark at room temperature for 3 hours. | Get A Quote |
In recent years， new labelling strategies have been developed that involve the genetic insertion of small amino-acid sequences for specific attachment of small organic fluorophores. Here， we focus on the tetracysteine FCM motif (FLNCCPGCCMEP)， which binds to fluorescein arsenical hairpin (FlAsH)， and the ybbR motif (TVLDSLEFIASKLA) which binds fluorophores conjugated to Coenzyme A (CoA) via a phosphoryl transfer reaction. We designed a peptide containing both motifs for orthogonal labelling with FlAsH and Alexa647 (AF647). Molecular dynamics simulations showed that both motifs remain solvent-accessible for labelling reactions. Fluorescence spectra， correlation spectroscopy and anisotropy decay were us... More
