Equivalent amounts of total HMC protein were added to buffer containing the LEHD caspase 9 peptide substrate linked to a cleavable luciferase substrate, aminoluciferin (Promega).
Since only chiA, and not chiB, is targeted by LhrA, we speculate that LhrA-regulation could be linked to a ChiA-specific substrate in the external environment, or possibly during infection, which would require differential regulation of the two genes.
We designed a new alkaline phosphatase (ALP -sensitive fluorogenic probe in which ALP -sensitiveive spacer group, p-hydroxybenzyl alcohol, is linked to a profluorogenic comprobe to inprove substrate specificity.
Commonly, Ub is linked to a lysine residue in the substrate protein, and since Ub itself has lysines on its surface, it can act as a target for Ub-ligases to produce poly-Ub chains.
Importantly, the fluorogenic substrates are linked to a quencher molecule and hence only become fluorescent after interacting with the target enzyme.
The enzymes characterized so far do not act on free substrates, but on substrate moieties which are covalently linked to a carrier-protein via a phosphopantetheinyl arm.
DOI: http://dx.doi.org/10.7554/eLife.05334.004 All native WaaL substrates contain carbohydrates linked to a common undecaprenyl (Und) lipid carrier.
Ubiquitination proceeds along an enzymatic cascade, in which ubiquitin is first activated by the ubiquitin-activating enzyme E1, conjugated by an E2 ubiquitin-conjugating enzyme, and finally covalently linked to the substrate by a specific E3 ubiquitin ligase [11],[11].
If we denote, at time t and pixel x, g x,t) as the grey level function, we can consider g as a food or substrate, which living entities (like bacteria) can eat, being attracted from the image boundaries (where they are first located) by a chemical gradient linked to the substrate.
The polydopamine film is loaded with a high density of anti-SRB antibodies linked to the substrate to obtain high response signals.
