Zenon Labeling Technology A New Approach to Immunolabeling from Molecular Probes

Transcription

1 Zenon Labeling Technology A New Approach to Immunolabeling from Molecular Probes Applications for Flow Cytometry and Imaging

2 A New Approach to Immunolabeling Zenon labeling technology provides a versatile, easy-to-use system for labeling mouse IgG 1, IgG 2a, IgG 2b and rabbit IgG antibodies with Molecular Probes premiere dyes as well as other fluorophore, biotin and enzyme labels. This new technology offers several advantages over direct chemical labeling, including: Speed The entire labeling procedure takes only 10 minutes. Efficiency Label nearly 100% of the primary antibody. Economy Label submicrogram amounts of antibody. Simplicity No pre- or post-labeling purification of the antibody is required. Flexibility Easily use multiple primary antibodies in a single experiment. Our wide selection of Zenon labeling reagents (Tables 1 and 2) can be mixed and matched, providing the freedom to experiment with multiple dye antibody combinations in flow cytometry and imaging applications. How Does the Technology Work? Zenon labeling technology uses a fluorophore-, biotin- or enzyme-labeled Fab fragment directed against the Fc portion of an intact IgG antibody to form a labeling complex (Figure 1, Figure 2). To ensure their high affinity and selectivity for the Fc portion of the target antibody, the Zenon labeling reagents have been affinity purified during their preparation. Because the Zenon labeling method is based on immunoselectivity, there is no need to remove exogenous proteins or amine-containing buffers from the antibody sample prior to forming the complex. Antibodies labeled using Zenon technology display fluorescence intensity or enzymatic activity similar to that observed for directly labeled antibody conjugates. A Unlabeled IgG antibody Zenon labeling reagent (labeled Fab fragment) Incubate B Labeled Fab fragments bound to the IgG C Mix with nonspecific IgG, which complexes unbound Fab fragments Figure 2. A ribbon diagram of the most likely configuration of Zenon Fab fragments bound to a mouse IgG 1 antibody. The antibody heavy and light chains are shown in blue and red, respectively. The Fab fragments (green) are shown bound at the junction between the CH 2 and CH 3 domains of the heavy chains, which are expected to be recognition sites for antibodies generated against the Fc portion of the molecule. Figure 1. The Zenon labeling scheme. An unlabeled IgG is incubated with the Zenon labeling reagent, which contains a fluorophore-labeled Fab fragment. The labeled Fab fragment binds to the Fc portion of the IgG antibody and excess Fab fragment is bound by the addition of a nonspecific IgG. The addition of nonspecific IgG prevents cross-labeling of the Fab fragment in experiments where multiple primary antibodies of the same type are present. Note that the Fab fragment used for labeling need not be coupled to a fluorophore, but could instead be coupled to an enzyme or to biotin.

3 Flow Cytometry Applications Zenon labeling technology is quick and convenient, and requires only 1 µg of primary antibody for the entire labeling procedure. The resulting Zenon staining complex is fully compatible with most flow cytometry applications and offers the following features: Suitable for multicolor applications (Figure 3) Brightness comparable to direct antibody conjugates (Figure 4) Compatible with live or fixed cultured cell samples Compatible with whole blood samples staining can be performed either before or after lysis of red blood cells Avoids the multiple steps involved with indirect staining procedures, especially in multicolor applications Eliminates the added time and expense of an Fc-blocking step Color combinations can be changed easily Figure 3. Human peripheral blood mononuclear cells stained with markers for CD3, CD4 and CD8 and detected using a lymphocyte gate. The cell sample was stained with an anti-cd3 mouse IgG 1 antibody prelabeled using the Zenon Alexa Fluor 488 Mouse IgG 1 Labeling Kit, an anti-cd4 mouse IgG 1 antibody prelabeled using the Zenon Alexa Fluor 647 R-Phycoerythrin Mouse IgG 1 Labeling Kit and an anti-cd8, mouse IgG 2a antibody prelabeled using the Zenon Alexa Fluor R-Phycoerythrin Mouse IgG 2a Labeling Kit. Plots of CD3 vs. CD4 staining (top), CD3 vs. CD8 staining (middle) and CD8 vs. CD4 staining (bottom) all demonstrate good signal separation and the expected differentiation of CD4+ and CD8+ cells. The samples were analyzed on a Coulter Elite flow cytometer using excitation at 488 nm and bandpass emission filters appropriate for the detection of Alexa Fluor 488 dye, R-phycoerythrin, and the Alexa Fluor 647 R-phycoerythrin tandem dye. Figure 4. Brightness comparison of phycoerythrin (R-PE, top) and allophycocyanin (APC, bottom) direct antibody conjugates with the comparable Zenon staining complexes. Human peripheral blood lymphocytes were incubated with a directly labeled antibody conjugate against the indicated CD marker according to the manufacturer s recommendations. In a parallel experiment, lymphocytes were incubated with 1 µg of a Zenon labeling complex formed using the unconjugated version of the same antibody, prepared according to the Zenon labeling kit protocol. In most cases, the Zenon staining complex produced an intensity that was comparable to or brigher than, that obtained from the direct conjugate. Note that the brightness of the Zenon staining complex can be further enhanced by increasing the ratio of Zenon labeling reagent to the primary antibody used in the preparation of the complex. Molecular Probes

4 Zenon Antibody Labeling Kits Table 1. Molecular Probes Zenon Labeling Kits. Label Abs/Em * Catalog Number Alexa Fluor Dyes Mouse IgG 1 Mouse IgG 2a Mouse IgG 2b Rabbit IgG Alexa Fluor /442 Z Z Z Z Alexa Fluor /539 Z Z Alexa Fluor /519 Z Z Z Z Alexa Fluor /554 Z Z Alexa Fluor /573 Z Z Z Z Alexa Fluor /565 Z Z Z Z Alexa Fluor /603 Z Z Z Z Alexa Fluor /617 Z Z Z Z Alexa Fluor /668 Z Z Z Z Alexa Fluor /690 Z Z Z Z Alexa Fluor /702 Z Z Z Z Alexa Fluor /719 Z Z Alexa Fluor /779 Z Z Classic Dyes Marina Blue 365/460 Z Z Cascade Blue 400/420 Z Pacific Blue 410/455 Z Z Fluorescein 494/518 Z Z Oregon Green /524 Z Z Texas Red-X 595/615 Z Z Biotins Biotin-XX NA Z Z Z Z DSB-X biotin NA Z Phycobiliproteins and Tandem dyes R-phycoerythrin (R-PE) 496 /578 Z Z Z Z Alexa Fluor 610 R-PE 496 /630 Z Alexa Fluor 647 R-PE 496 /668 Z Z Z Alexa Fluor 680 R-PE 496 /702 Z Allophycocyanin (APC) 650/660 Z Z Z Z Alexa Fluor 700 APC 650/723 Z Alexa Fluor 750 APC 650/775 Z Enzymes Horseradish peroxidase NA Z Z Z Z Alkaline phosphatase NA Z Z Z Z * Approximate absorption and emission maxima, in nm. Each Zenon Labeling Kit with the Alexa Fluor dyes, classic dyes or biotins contains materials for 50 labelings; one labeling is defined as the amount of Zenon reagent required to label 1 µg of antibody. Each Zenon Labeling Kit with phycobiliproteins or enzymes contains materials for 25 labelings; kits with tandem dyes contain materials for 10 labelings. One labeling is defined as the amount of Zenon reagent required to label 1 µg of antibody. Additional absorption peaks are present at 546 and 565 nm. NA = Not applicable. Call or check our Web site for current pricing. Table 2. Molecular Probes Zenon Tricolor Labeling Kits. Tricolor Labeling Kit Labels * Catalog Number Kit #1 (for imaging) Kit #2 (for imaging) Kit #3 (for flow cytometry) Alexa Fluor 488 Alexa Fluor 555 Alexa Fluor 647 Alexa Fluor 350 Alexa Fluor 488 Alexa Fluor 594 Alexa Fluor 488 R-phycoerythrin (R-PE) Alexa Fluor 647 R-PE Mouse IgG 1 Mouse IgG 2a Mouse IgG 2b Rabbit IgG Z Z Z Z Z Z Z Z Z Z Z * See Table 1 for absorption and emission maxima. Each Zenon Tricolor Labeling Kit contains materials for 10 labelings with each of the three included labeling reagents. Call or check our Web site for current pricing.

5 Imaging Applications Zenon labeling technology simplifies time-consuming immunocytochemical applications such as the use of multiple same-species derived primary antibodies in the same staining protocol (Figures 5 8). The extensive selection of available labels and the versatility of Zenon technology makes it easy to experiment with different color combinations for optimal multicolor images. More examples of Zenon technology in action can be seen in the gallery section of our Web site ( Figure 5. An MRC5 fibroblast labeled with probes for mitochondria and the phosphorylated form of histone H3. Mitochondria were detected with an anti pyruvate dehydrogenase mouse IgG 2a monoclonal antibody prelabeled using the Zenon Alexa Fluor 488 Mouse IgG 2a Labeling Kit, and phosphorylated histone H3 was detected with an anti phosphohistone H3 rabbit IgG antibody prelabeled using the Zenon Alexa Fluor 555 Rabbit IgG Labeling Kit. The nucleus was counterstained with DAPI. Figure 6. A431 cells labeled with probes for histones and the epidermal growth factor (EGF) receptor. Histones were detected with an anti-histone mouse IgG 2a monoclonal antibody prelabeled using the Zenon Alexa Fluor 488 Mouse IgG 2a Labeling Kit. EGF receptors on the cell surface were detected with an anti EGF receptor mouse IgG 2a monoclonal antibody prelabeled using the Zenon Alexa Fluor 555 Mouse IgG 2a Labeling Kit. Figure 7. Bovine pulmonary artery endothelial cells labeled with probes for actin, histones, and the mitochondria. Histones were detected with an anti-histone mouse IgG 2a monoclonal antibody prelabeled using the Zenon Alexa Fluor 488 Mouse IgG 2a Labeling Kit, and mitochondria were detected with an anti pyruvate dehydrogenase mouse IgG 2a monoclonal antibody prelabeled using the Zenon Alexa Fluor 555 Mouse IgG 2a Labeling Kit. Actin was labeled with Alexa Fluor 350 phalloidin. Figure 8. Bovine pulmonary artery endothelial cells labeled with probes for the cytoskeleton, mitochondria, and the phosphorylated form of histone H3. The cytoskeleton was detected with an anti-vimentin mouse IgG 1 monoclonal antibody prelabeled using the Zenon Alexa Fluor 488 Mouse IgG 1 Labeling Kit, and mitochondria were detected with an anti pyruvate dehydrogenase mouse IgG 2a monoclonal antibody prelabeled using the Zenon Alexa Fluor 555 Mouse IgG 2a Labeling Kit. Phosphorylated histone H3 was detected with an anti phosphohistone H3 rabbit IgG antibody prelabeled using the Zenon Alexa Fluor 350 Rabbit IgG Labeling Kit.

6 Molecular Probes, Inc Willow Creek Rd. Eugene, OR Phone: (541) Fax: (541) Customer Service Phone: (541) Fax: (541) For US and Canada Toll-Free Order Phone: (800) Toll-Free Order Fax: (800) Technical Assistance Phone: (541) Fax: (541) Madin-Darby canine kidney (MDCK) cells showing the distribution of the tight junction protein ZO-3 and mitochondria. ZO-3 was detected with a rabbit anti ZO-3 antibody prelabeled using the Zenon Rabbit Alexa Fluor 488 IgG Labeling Kit. Endogenous biotin in the mitochondria was detected with streptavidin and visualized with a rabbit antistreptavidin antibody prelabeled using the Zenon Rabbit Alexa Fluor 555 IgG Labeling Kit. The cells were counterstained with DAPI to mark the nucleus. Molecular Probes Europe BV PoortGebouw, Rijnsburgerweg AA Leiden, The Netherlands Phone: Fax: Customer Service Phone: Fax: eurorder@probes.nl Technical Assistance Phone: Fax: eurotech@probes.nl All digital fluorescence microscopy images contributed by Molecular Probes scientists were acquired using CCD cameras controlled by MetaMorph software (Universal Imaging Corporation, The manufacture, use, or sale of the products in this brochure may be covered by one or more of the following U.S. patents: 5,132,432; 5,696,157; 5,798,276; 5,830,912; 5,955,612; 6,130,101; 6,162,931; 6,229,055; or one or more foreign patents, or pending U.S. or foreign patent applications. For research use only. Alexa Fluor, Cascade Blue, Marina Blue, Oregon Green and Texas Red are registered ( ) trademarks of Molecular Probes, Inc. DSB-X, Pacific Blue and Zenon are trademarks ( ) of Molecular Probes, Inc. TC0258-1