Zenon labeling technology provides a rapid, versatile, and reliable method for producing fluorescently labeled antibodies using a variety of fluorochromes, even with small amounts of unpurified starting material such as hybridoma culture supernatant. Antibodies labeled using Zenon labeling technology are suitable for use in all applications where a directly labeled antibody can be used. Advantages of Zenon labeling technology include:
- 10 minute labeling time
- 1–20 µg primary antibody per labeling
- Compatible with BSA and other stabilizing proteins
- No purification step
- Flexible antibody labeling
How Zenon labeling technology works
Zenon labeling technology provides a versatile, easy-to-use system for labeling human, mouse IgG1, IgG2a, and IgG2b antibodies and rabbit IgG antibodies. Zenon® fragments are specifically designed to target and bind to the Fc portion of the primary antibody only, giving a rapid, noncovalent method of quickly labeling small quantities of primary antibody (Figure 1). Zenon labeling technology is simple and efficient; the entire labeling procedure takes only 10 minutes.
Once the Zenon–antibody complex has formed, it can be stored until needed; just prior to use, nonspecific IgG is added to block any unbound Zenon fragments, making column purification unnecessary. The Zenon complex can then be applied directly to the sample.
Antibodies labeled using Zenon labeling technology display fluorescence intensity or enzymatic activity similar to that observed for directly labeled antibody conjugates (Figure 2).
Figure 2. Zenon Labeling Technology produces antibody conjugates with brightness comparable to or better than those obtained from direct conjugates. Antibodies to various lymphocyte markers were labeled either covalently with allophycocyanin (APC) or noncovalently with 1 μg of a Zenon APC complex. Labeled antibodies were then used to stain samples of human peripheral blood lymphocytes. The brightness of the Zenon staining complex can be further enhanced by increasing the ratio of Zenon labeling technology reagent to the primary antibody used in the preparation of the complex.
Ultimate flexibility with Zenon Antibody Labeling Kits
Zenon labeling technology offers unique flexibility for when you want to change colors or test whether a particular fluorophore is suitable for your application or antibody. The extensive selection of labels and the versatility of this labeling technology make it easy to experiment with different color combinations for multicolor applications in both flow cytometry (Figure 3) and fluorescence imaging (Figures 4 and 5).
Figure 3. Use of Zenon Labeling Technology in flow cytometry. Human peripheral blood lymphocytes were stained with the following three antibodies: an anti-CD3 mouse IgG1 antibody prelabeled with the Zenon Alexa Fluor 647 Mouse IgG1 Labeling Kit (Z25008), an anti-CD4 mouse IgG1 antibody prelabeled with the Zenon R-Phycoerythrin Mouse IgG1 Labeling Kit (Z25055) and an anti-CD8 mouse IgG2a antibody prelabeled with the Zenon Alexa Fluor488 Mouse IgG2a Labeling Kit (Z25102). Panels A and B show that cells can be separated by plotting the orange-fluorescent versus green-fluorescent signal or red-fluorescent versus orange-fluorescent signal, respectively, demonstrating that Zenon labeling technology does not transfer to other antibodies in the same sample. The samples were analyzed on a Coulter Elite flow cytometer using 488 nm excitation for R-phycoerythrin and the Alexa Fluor 488 dye, and 633 nm excitation for the Alexa Fluor 647 dye.
Figure 4. Use of Zenon Labeling Technology in fluorescence imaging. A 14 µm coronal section of mouse hippocampus stained with an anti–α-tubulin antibody (A11126) labeled with the Zenon Alexa Fluor 488 Mouse IgG1 Labeling Kit (Z25002). The section was counterstained with the NeuroTrace 530/615 red fluorescent Nissl stain (N21482) to visualize neuronal cell bodies and Hoechst 33258 (H1398, H3569, H21491) to stain nuclei.
Figure 5. Use of Zenon Labeling Technology in fluorescence imaging. Bovine pulmonary artery endothelial cells labeled with probes for tubulin and the mitochondria. Tubulin was detected with an anti–α-tubulin mouse IgG2b monoclonal antibody prelabeled with the Zenon Alexa Fluor 488 Mouse IgG2b Labeling Kit (Z25202), and mitochondria were labeled using an anti-OxPhos Complex V subunit α, mouse IgG2b monoclonal antibody prelabeled with the Zenon Alexa Fluor 555 Mouse IgG2b Labeling Kit (Z25205). The nucleus was stained with DAPI (D1306, D3571, D21490).