Labeling proteins

Qualmann and Kessels have reported the synthesis of carborane-containing lysine dendrimers (123) (Fig. 72), with a better defined number of boron atoms, for use as protein labels in immunocytochemistry using electron microscopic techniques such as electron energy loss spectroscopy (EELS) and electron spectroscopic imaging (ESI).149... 

Wu XL, Tian M, Fie HZ et al (2009) Synthesis and biological applications of two novel fluorescent proteins-labeling probes. Bioorg Med Chem Lett 19 2957-2959... 

Adams SR, Campbell RE, Gross LA et al (2002) New Biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo synthesis and biological applications. J Am Chem Soc 124 6063-6076... 

Yan W, Sloat AL, Yagi S, Nakazumi H, Colyer CL (2006) Protein labeling with red squarylium dyes for analysis by capillary electrophoresis with laser-induced fluorescence detection. Electrophoresis 27 1347-1354... 

Oswald B, Patsenker L, Duschl J, Szmacinski H, Wolfbeis OS, Terpetschnig E (1999) Synthesis, spectral properties, and detection limits of reactive squaraine dyes, a new class of diode laser compatible fluorescent protein labels. Bioconjugate Chem 10 925-931... 

Oswald B, Gruber M, Bohmer M, Lehmann F, Probst M, Wolfbeis OS (2001) Novel diode laser-compatible fluorophores and their application to single molecule detection, protein labeling and fluorescence resonance energy transfer immunoassay. Photochem Photobiol 74 237-245... 

Umezawa K, Citterio D, Suzuki K (2008) Water-soluble NIR fluorescent probes based on squaraine and their application for protein labeling. Anal Sci 24 213-217... 

In one set of experiments a titration of compound is performed to assess its potency in vivo. HeLa cells are maintained in DMEM supplemented with 10% fetal bovine serum (FBS) at 37° in 5% C02. One day prior to labeling, the cells are seeded in 24-well plates at approximately 60,000 cells per well. The next day, cells are washed with warm (37°) PBS and the medium replaced with 250 41 of methionine-free DMEM containing 10% dialyzed serum (Invitrogen). After a 15-min incubation at 37°, different concentrations of compound are added to the cells (which can range from 1 nM to 50 fiM) and the incubation continued for another 45 min. Anisomycin is used as a positive control at a final concentration of 50 /iM. Fifty-five microcuries of 35S-methionine/cysteine [35S-methionine/cysteine express protein labeling mix (1175 Ci/mmol) (Per-kin-Elmer)] is added to each well (220 /(Ci/ml) and the incubation continued for another 15 min. 

Nonaka, H., Tsukiji, S., Ojida, A. and Hamachi, I. (2007). Non-enzy-matic covalent protein labeling using a reactive tag. J. Am. Chem. Soc. 129, 15777-9. 

AMCA-NHS, succinimidyl-7-amino-4-methylcoumarin-3-acetic acid, is an amine-reactive derivative of AMCA containing an NHS ester on its carboxylate group (Thermo Fisher). The result is reactivity directed toward amine-containing molecules, forming amide linkages with the AMCA fluorophore (Figure 9.23). Proteins labeled with AMCA show little-to-no effect on the isoelectric point of the molecule. 

Cyanine dyes also are used as labels for oligonucleotide probes. Unlike the hydrophilic cyanine dyes valuable for protein labeling, the use of dye-phosphoramidite compounds to synthesize DNA or RNA probes typically requires the use of more hydrophobic dye structures to make them compatible with the solvents and reactions of oligonucleotide synthesis. Thus, indol cyanines containing few or no sulfonates are used in these applications to label oligos for applications such as array detection, hybridization assays, and RT-PCR. 

Preparation of Mono-Disperse Gold Suspensions for Protein Labeling... 

Protein labeled with iron chelate groups via thioether linkage... 

Analyze the cleaved proteins by SDS gel electrophoresis and Western blotting, followed by specific detection of the prey protein label. Comparison of prey protein fragments formed by chemical and enzymatic digestion can yield information as to the site of the resultant FeBABE-mediated cleavage. 

Lacey, B., and Grant, W.N. (1987) Photobiotin as a sensitive probe for protein labeling. Anal. Biochem. 163, 151-158. 

Qualmann, B., Kessels, M.M., Musiol, H.J., Sierralta, W.D., Jungblut, P.W., and Morodeg L. (1996) Synthesis of boron-rich lysine dendrimers as protein labels in electron microscopy. Angew. Chem. Int. 

Schwartz, D.A., Abrams, M.J., Giadomenico, C.M., and Zubieta, J.A. (1995) Protein labelling utilizing certain pyridyl hydrazines, hydrazides and derivatives. US Patent No. 5,420,285. 

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