In-gel fluorescence

Gel-based ABPP enables visualization of labeling events using gel electrophoresis accompanied by in-gel fluorescence (rhodamine) or... 

Fig. 4 Identification of alkyne-tagged P-lactone probes that target ClpP in S. aureus using click-chemistry, in-gel fluorescence and mass spectrometry. Subsequently, lead compounds were administered to 5. aureus and changes to the global bacterial proteome monitored. Particularly, changes to the levels of haemolysis and proteolysis enzymes (haemolysins, proteases, lipases and...

In P. aeruginosa, despite considerably lower levels of LasR when compared to the E. coli system, the same protocol resulted in detection of a band by in-gel fluorescence, which could be excised and the identification of LasR achieved by proteomic MS methods with 32% total sequence coverage. The methodology was also extended to allow analysis by flow cytometry. In this way, the authors were able to demonstrate that cell density had an effect on LasR labelling. As expected, fluorescence increased with cell density up to the point at which quorum was achieved (around OD600 = 6), beyond which fluorescence began to weaken. 

Figure 4 Comparative profiling of enzymatic activity states in the proteomes of nonpathogenic and pathogenic origin. Disease-associated targets can be determined based on the activity pattern revealed by analytical methods such as SDS gel electrophoresis coupled with in-gel fluorescence scanning. Reproduced by the permission of The Royal Society of Chemistry.

S. Robia, N. Flohr, D. Thomas, Phospholamban pentamer quaternary conformation determined by in-gel fluorescence anisotropy, Biochemistry 2005, 44,4302-4311. 

Fig. 1. Schematic of the method for monitoring myristoylated and palmitoylated cellular proteins, (a) co-Alkynyl fatty acid probes for detecting protein myristoyiation and paimitoyiation. (b) (o-Alkynyl fatty acids added to growth media are metabolically incorporated into cellular fatty-acylated proteins. The cells are then lysed, Click chemistry is used to chemoselectively conjugate a biotin-azide or rhodamine-azide to fatty-acylated proteins, and the proteome is detected by western blotting or in-gel fluorescence. Alk-CI 3 and Alk-Cf4 are probes for detecting protein myristoyiation, while Alk-CI6 and Alk-CfS detect protein paimitoyiation.

The user can develop the signal by using rhodamine-azide followed by in-gel fluorescence detection. This yields minimal background and high signal-to-noise ratio compared to biotin-azide. Label cells and run gel, but replace biotin-azide with rhodamine-azide in Subheading 3.5 step 2. 

Clark PM, Dweck JF, Mason DE, Hart CR, Buck SB, Peters EC, Agnew BJ, Hsieh-Wilson LC (2008) Direct in-gel fluorescence detection and cellular imaging of O-GlcNAc-modified proteins. J Am Chem Soc 130 11576-11577... 

Wittig, I., Karas, M., and Schagger, H. (2007) High resolution clear native electrophoresis for in-gel functional assays and fluorescence studies of membrane protein complexes. Mol. Cell. Proteomics 6, 1215-1225. 

Figure 11.3 Transmitted polarized light images of a thermally oxidized and crosslinked gel in a multilayer film a) photograph in polarized light, and b) the gel fluorescing under UV light. Photographs were provided by E. Garcia-Meitin of The Dow Chemical Company...

Compound Structure formula Posit, of fluoresc. max. of vapor, mjTb Fluoresc. quench, in vapor Fluoresc. quench, in adsorb, on silica gel... 

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