The advantages of tandem dyes

A significant number of laboratory applications rely on the use of fluorescently-labeled antibodies. These reagents are ideally suited to multiplexing experiments, since by labeling antibodies against different protein targets with different colored dyes, it is possible for several readouts to be obtained from a single precious sample.

One technique which utilizes fluorometric detection is flow cytometry, an application which analyzes the characteristics of cells as they flow singly past beams of focused light. The cells can be separated in to different populations based on light scatter, and can also be sorted on the basis of protein expression; it is the latter which requires the use of fluorescently-labeled antibodies.

During fluorometric detection the fluorophore that is bound to the antibody becomes excited by the light source. It then produces a transient light emission as it returns to its ground state, generating light at a higher wavelength than that which was used for excitation. The difference between the maximum absorbance and emission wavelengths of the fluorophore is defined by the Stokes shift (see image below). A large Stokes shift is indicative of less overlap between the two wavelengths, and is highly desirable when performing flow cytometry since it means that the emitted light can be clearly distinguished from the light source which was used for excitation.

Although antibodies can be labeled with a single species of fluorophore, it is possible to exploit the Stokes shift through the use of tandem dyes. These consist of two covalently attached fluorophores, one of which behaves as a donor molecule while the other acts as an acceptor molecule. Once the donor molecule has been excited by the light source, a proportion of its energy is transferred to the acceptor molecule via Fluorescence Resonance Energy Transfer. This excites the acceptor molecule, which in turn produces a light emission. The Stokes shift of a tandem dye is greater than that of the original donor molecule. Another benefit of tandem dyes is that they can be used to increase the quantity of readouts from a flow cytometry experiment should the number of excitation lasers be a limiting factor. For example, DyLight® 488, R-phycoerythrin (PE), PerCP/Cy5.5 and PE/Atto 594 can all be excited using a 488nm laser, yet generate green, yellow, red and infra-red emissions respectively, each of which can be measured using a different detector.

As the number of assay readouts increases, it becomes necessary to use directly-labeled primary antibodies, rather than labeled secondary antibodies, for detection. The reason for this is that the use of secondary antibodies prohibits the use of more than one primary antibody from the same host species in the experiment.

direct vs indirect detection - immunofluorescence

Direct vs. indirect staining. The image on the left illustrates the detection of three different cellular proteins using directly-labeled primary antibodies; each protein is clearly defined by a different colored fluorochrome. The image on the right illustrates the detection of the same three cellular proteins using a labeled secondary antibody; since all three primary antibodies share the same host species, differential staining on the basis of color is not possible.

Unfortunately it is often the case that fluorescently-labeled versions of the primary antibodies are not commercially available, however we’ve solved this problem by developing our Lightning-Link® antibody labeling kits. These easy-to-use kits allow direct conjugation of primary antibodies to fluorescent labels, including tandem dyes, with just 30 seconds hands-on time and a 100% yield from the unconjugated antibody. Simply pipette your antibody into a vial of lyophilized mixture containing the label of interest, incubate, and then stop the reaction. With no desalting or dialysis steps, your conjugate is then ready to use. View the video protocol demonstration here:

We have almost 40 fluorescent dyes in our expanding Lightning-Link® range, of which eight are tandem dyes:

Tandem dyes table

The PE/Atto594 tandem dye is unique to Innova Biosciences. It is brighter, and has a greater Stokes shift, than the more commonly-used PE/Texas Red® tandem, and has been literature cited. In their article “Using Image-Based Flow Cytometry to Assess Monocyte Oxidized LDL Phagocytosis Capacity”, published in Methods in Molecular Biology, Henning et al. describe the use of Lightning-Link® PE/Atto594 to label an anti-CD16 antibody within a study to evaluate how acute dietary modifications can alter the risk of developing atherosclerosis.

Comparison of the absorbance and fluorescence emission spectra of Lightning-Link® PE/Atto594 and Lightning-Link® PE/Texas Red® conjugates

Comparison of the absorbance and fluorescence emission spectra of Lightning-Link® PE/Atto594 and Lightning-Link® PE/Texas Red® conjugates. Although both tandem dyes share similar profiles, PE/Atto594 is significantly brighter, and has a greater Stokes shift, than PE/Texas Red®.

View all of our Lightning-Link® antibody labeling kits here. For more information on multi-color flow cytometry, take a look at our Beginner’s Guide to Flow Cytometry, or visit our multi-color flow cytometry page.