Fluorescence labelling of biological molecules

Introduction

The fluorescent assay reagent toolbox has evolved from a class of dyes referred to as the cyanines which are characterized by a resonance structure, bearing an overall single positive charge. Amersham Biosciences CyDye^TM fluorophores were pioneered by Professor Alan Waggoner and are licensed from Carnegie Mellon University, Pittsburgh. CyDye fluors are particularly suited as fluorescent probes since their red to near infrared absorption and emission maxima (500-800 nm) are easily distinguishable from the shorter wavelength endogenous autofluorescence associated with biological samples and equipment. Furthermore, they have been designed to take full advantage of LEADseeker^TM Homogenous Imaging System since the CCD chip is most sensitive to light in the red region of the spectrum. Additional characteristics of CyDye fluorophores include relatively narrow bandwidths, large molar extinction coefficients (the light absorbing capability of a fluor), good aqueous solubility and photostability, low non-specific binding properties, favourable fluorescent quantum yields (the efficiency with which a fluor converts absorbed light to emitted fluorescent light) and pH insensitivity. This composite of properties enables the development of robust, sensitive assays.

N-hydroxysuccinimide (NHS) esters

Perhaps the most convenient and widely used functional group for the labelling of biological molecules is a primary amino group. This can be provided by the e-amino group of a lysine residue, or the free N-terminus of a peptide/protein. Alternatively, it is possible to introduce primary amine containing modifier groups during automated synthesis of, for example, oligonucleotides. Stable active esters of fluor labelling species that may be stored as solid materials, in particular NHS esters (including for example CyDye NHS reagents), have been extensively used over many years for the acylation of such amino groups, as shown in the reaction schematic below.

At Amersham Biosciences, this approach has been utilized to label a number of molecules such as peptides, oligonucleotides and proteins/antibodies, which have subsequently been successfully employed in a range of fluorescent application assays.

Maleimides

As an alternative to primary amino labelling, thiol containing groups such as those contained in cysteine residues or, as with primary amino groups, those introduced as modifiers during automated synthesis (of for example oligonucleotides) can be specifically targeted by maleimide labelling reagents (for example of Cy3 and Cy5) as shown in the reaction schematic below.

Hydrazides

It is not always feasible to consider the use of either primary amino or thiol labelling on all biological molecules. A further common route for labelling of, for example, carbohydrate species is via an aldehyde group targeted by hydrazide labelling reagents (for example of Cy3 and Cy5) as shown in the reaction schematic below.

Other labelling reagents

Amersham Biosciences also provides additional reagents through its catalog range. These include Cy3 and Cy5 phosphoramidites for direct labelling of oligonucleotides during synthesis and kits designed and optimized specifically for CyDye labelling of antibodies, together with detailed protocols on their use

Here is a table that allows you to calculate your dye - protein ratio

Biological labelling service

Amersham Biosciences also offers a Fluorescence Labelling Service. This is a dedicated custom labelling service for the preparation of CyDye labelled products, typically peptides, proteins and DNA. Customers are able to take advantage of our expertise in custom synthesis and labelling, assay design and exclusive CyDye reagents in order to achieve optimal substrate or ligand labelling. Techniques available to the Fluorescence Labelling Service include, specific positional labelling of peptides (mono-labelled and FRET pairs), specific functional group labelling of proteins and site specific labelling of oligonucleotides.

Please contact your local area representative for more details.