Fluorescent Chromeo™ Py-Dyes

Pyrylium dyes change their color and fluorescent properties upon reacting with proteins. After binding to primary amines, they undergo a large shortwave spectral shift that is visible to the naked eye (Figure 1). There is also a large increase in quantum yield that changes the non- or only weakly fluorescent dye into a bright fluorescent conjugate. (See the Properties tab for detailed information on each Py-Dye).

Figure 1: Color shift of Chromeo Py-Dyes caused by dye conjugation.
Chromeo P503 is blue while in its free state. After the short conjugation reaction, the dye's color undergoes a large Stokes shift and becomes red.

The shift of the absorption & emission bands together with the increased fluorescence quantum yield effectively eliminates background from any unbound dye. In addition, because unbound pyrylium dye is hydrolyzed during the labeling procedure, there is no need for purification following the simple one-step, room-temperature incubation. The labeled protein is ready to use immediately, and any possible background from unbound dye is eliminated. Due to the unique conjugation mechanism, proteins labeled with Py-Dyes maintain their native charge and isoelectric point. This is an important improvement over other derivatization-based methods for labeling proteins because it eliminates band or peak broadening in different assays and maintains the native characteristics of the proteins.

Chromeo™ Py-Dye advantages

• No washing steps required
• No background from unbound dye
• Fast and simple procedure
• Small size
• No change in net charge of the labeled molecule
• No interference of excitation and emission wavelength

Because of their unique features, Chromeo Py-Dyes (Chromeo P429, P503, P540 and P543) have been used successfully in a number of “no-wash” applications such capillary electrophoresis, SDS-protein gel electrophoresis, isoelectric focusing or as a label in receptor binding studies. See the Product Citations tab for a sample of published references.

Published references of Py-Dye applications

Pre-stain in SDS-gel electrophoresis

Amine-reactive pyrylium dyes have been used as a pre-stain in protein electrophoresis. The high sensitivity with a detection-limit of 16 picograms and the fast, reproducible procedure improve electrophoresis results compared to other commercially available stains like SYPRO Red or silver staining.

“SDS-PAGE of proteins using a chameleon-type of fluorescence prestain” by Meier et al (2008) Anal. Chemistry 80(16):6274-6279.
 
“Gene optimization mechanisms: A multi-gene study reveals a high success rate of full-length human proteins expressed in Escherichia coli” by Maertens et al (2010) Protein Science 19:1312-1326.

Protein stain in capillary electrophoresis (CE)

Pyrylium dyes are useful as a stain in capillary electrophoresis. The detection limit in the picomolar range, the fast reaction time and the preservation of the protein’s native charge make Chromeo Py-Dyes an interesting alternative to commonly used detection techniques.

“Determination of picomolar concentrations of protein using novel amino reactive chameleon labels and capillary electrophoresis laser-induced fluorescence detection” by Craig et al (2005) Electrophoresis 26(11):2208-2213.
 
“Reaction of fluorogenic reagents with proteins: II. Capillary electrophoresis and laser-induced fluorescent properties of proteins labeled with Chromeo P465” by Swearingen et al (2008) J. of Chromatography 1194:249-252.
 
“Reaction of organic reagents with proteins III. Spectroscopic and electrophoretic behavior of proteins labeled with Chromeo P503” by Turner et al (2008) J. of Chromatography 1194(2):253-256.
 
“Determination of biogenic amines by capillary electrophoresis using a chameleon type of fluorescent stain” by Steiner et al (2009) Microchimica Acta 167 (3-4):259-266.
 
“Capillary Electrophoresis” by Frost et al (2010) Anal. Chem 82 (12):4682-4698.
 
“Protein labeling Enhances Aptamer Selection by Methods of Capillary Electrophoresis” by de Jong et al (2011) Anal. Chem. 83:6330-6335.
 
“Preasure-Based Approach for the Analysis of Protein Adsorption in Capillary Electrophoresis; by de Jong et al (2012) Anal. Chem. 84:453-458.
 
“On-capillary fluorescent labeling and CE-LIF analysis of glycoforms of intact prostate-specific antigen” by Garrido-Medina et al (2013) Electrophoresis 34 (16):2295-2302.

Stain in microchip applications

On- and off-chip labeling on miniaturized platforms demonstrates the use of pyrylium dyes in the development of detection methods that require small amount of sample and feature fast analysis times.

“Polymer microchip CE of proteins either off- or on-chip labeled with chameleon dye for simplified analysis” by Yu et al (2009) Electrophoresis 30(24):4230-4236.
 
“Development of an SDS-Gel Electrophoresis method on SU-8 Microchips for protein separation with laser-induced fluorescence detection: Application to the analysis of WHEY proteins” by del Mar Barrios-Romero et al (2013) J. of Separation Science 36(15):2530-2537.

Protein stain in capillary isoelectric focussing (CIEF)

Pyrylium dyes are excellent stains in capillary isoelectric focussing as they preserve the isoelectric point of the labeled proteins.

“Attomole protein analysis by CIEF with LIF detection” by Ramsay et al (2009) Electrophoresis 30(2):297-302.
 
“Femtomolar Concentration Detection Limit and Zeptomolar Mass Detection Limit for Protein Separation by Capillary Isoelectric Focussing and Laser-Induced Fluorescence Detection” by Ramsay et al (2009) Anal.Chem. 81(5):1741-1746.
 
“Capillary array isoelectric focussing with laser-induced fluorescence detection: milli-pH unit resolution and yoctomole mass detection limits in a 32-channel system” by Dada et al (2010) Anal. Bioanal. Chem. 397(8):3305-3310.
 
“Two-dimensional capillary electrophoresis: Capillary isoelectric focusing and capillary zone electrophoresis with laser-induced fluorescence detection” by Dickerson et al (2010) Electrophoresis 31:2650-2654.

Label for carbohydrate characterization

Chromeo P503 (Py-1) was used to label a mixture of ethylenediamine functionalized oligosaccharides. The fluorescent carbohydrates have been analyzed in capillary electrophoresis (CE) and mass spectrometry (MALDI-ToF-MS).

“Glycan analysis via derivatization with a fluorogenic pyrylium dye” by Johannesen et al (2012) Carbohydrate Research 352:94-100.

Small protein label for functional analysis

Due to its small size, Chromeo P503 (Py-1) was used to fluorescently label a set of NPY receptor antagonists. The functionality of the labeled structures was shown by flow cytometry and confocal microscopy.

“Synthesis and Characterization of the First Fluorescent Nonpeptide NPY Y1 Receptor Antagonist” by Schneider et al (2007) ChemBioChem 8(16):1981-1988.
 
“Red-fluorescent argininamide-type NPY Y1 receptor antagonists as pharmacological tools” by Keller et al (2011) Bioorganic & Medical Chem. 19(9):2859-2878.

Monitoring of amino functionalities

Chromeo P503 (Py-1) was used to screen and evaluate the surface of polymer films by fluorescence spectroscopy and confocal scanning microscopy. The spectral shift of the bound dye enabled the distinction between covalently bound and nonspecifically adsorbed dye.

“Monitoring of Amino Functionalities on Plasma-Chemically Modified Polypropylene Supports with a Chromogenic and Fluorogenic Pyrylium Reporter” by Hoffmann et al (2007) Langmuir 23(16):8411-8416.
 
“Fluorescence measurements on Functionalized Polymer Surfaces-Problems and Troubleshooting” by Hoffmann et al (2008) Ann. N.Y. Acad. Sci. 1130:28-34.

General application

“New Approach for Fluorescent Peptide Labeling with Pyrylium Cyanine dyes” by Kostenko et al (2002) J. of Fluorescence 12(2):173-175.
 
“Chameleon Labels for Staining and Quantifying Proteins” by Wetzel et al (2004) Angew. Chemie Int. Ed. 43:5400-5402.
 
“Novel type of general protein assay using a chromogenic and fluorogenic amine-reactive probe” by Hoefelschweiger et al (2005) Analytical Biochem. 344:122-129.
 
“Screening Scheme Based on Measurement of Fluorescence Lifetime in the Nanosecond Domain” by Wetzel et al (2005) J. Biological Screening 10:685-694.
 
“Reaction of fluorogenic reagents with proteins: I. Mass spectrometric characterization of the reaction with 3-(2-furoyl)quinoline-2-carboxaldehyde, Chromeo P465 and Chromeo P503” by Wojcik et al (2008) Journal of Chromatography 1194:243-248.
 
“Chromogenic Sensing of Biogenic Amines using a Chameleon Probe and the Red-Green-Blue Readout of Digital camera Images” by Steineret al (2010) Analytical Chemistry 82(20):8402-8405.
 
“High-throughput sensing microtiter plate for determination of biogenic amines in seafood using fluorescence or eye-vision” by Azab et al (2011) Analyst 136:4492-4499.

Contents & Storage

Each Chromeo Py-Dye is supplied as a dry powder. Guaranteed stable for 6 months when stored properly.