Convenient analysis of protein modification by chemical blotting with fluorogenic “click” reagents

Abstract

Direct visualization of bioorthogonal alkyne or azide handles using fluorogenic azide-alkyne cycloaddition conducted on the surface of a blot membrane. The method eliminates the need for separation steps to remove excess small molecule reagents before attachment of antigen molecules or other visualization handles, and is especially useful for the analysis of peptides and small proteins. A variety of potential fluorogenic reagents are assessed, and sensitivity (<0.1 picomole) similar to current commercially available fluorescence imaging methods is possible.

A Three‐Component Organometallic Tyrosine Bioconjugation

“…We conducted a broad screen of transition‐metal salts for modification with an alkyne‐functionalized arylboronic acid, using “chemical” western blot analysis, to identify covalent incorporation of the boronic acid group, through visualization of the pendant alkyne. This screen of many transition metals uncovered an alkyne‐tagged protein for only two metals: the copper(II)‐dependent process previously reported as a Chan–Lam coupling, and a heretofore unknown process involving rhodium.…”

Development of a 1,2-difluorofucoside activity-based probe for profiling GH29 fucosidases

“…This method has previously been shown to have similar sensitivity compared to regular imaging methods and it eliminates the need for labor intensive separation steps to remove excess of probe before attachment of the visualization handle. 46 Incubation of YL209 with BfFucH followed by SDS-PAGE and Western Blot analysis revealed no signal when normal electrophoresis protocols were followed. It has previously been discussed in literature that this result can occur due to hydrolysis of the ester linkage of the glycosyl-enzyme intermediate under the basic conditions ( pH 8.8) of the buffered gel.…”

A Naturally Encoded Dipeptide Handle for Bioorthogonal Chan–Lam Coupling

“…Theboronic acid coupling can be used to introduce useful functionality and other uniquely reactive handles.W esynthesized trifunctional boronate reagent 2,c ontaining ad esthiobiotin [23] affinity handle and an azide group on ac ysteine (Figure 4d). Thec opper modification reaction and affinity purification of Glp-His-tagged GFP was performed with reagent 2,a nd incorporation of an azide handle was confirmed by direct detection of the azide group on ab lot membrane [24] (Figure 4e," chem blot"). GFP modified with 1b lacking the azide group gave no such fluorescence ( Figure S11).…”