New Simplified Workflow Options for Maximum-Yield Protein Conjugations

From APEX® to Zenon®: Molecular Probes® Protein Labeling Kits

Take Advantage of Our Protein Conjugation Experience

Table 1 shows a summary of Molecular Probes® protein labeling kits—including the APEX® and Zenon® antibody labeling kits and our range of Alexa Fluor® protein labeling kits—with emphasis on the different starting material requirements and protocols for each kit. This selection guide will point you to the kits best suited for your experiments, from microgram- to milligram-scale protein labeling. For each kit format, we offer a choice of dye and hapten labels.

We also offer custom conjugation services for any project, large or small. We have considerable expertise in synthesizing and labeling biologically relevant molecules for use as detection reagents and site-selective probes. Please email us at custom.services@lifetech.com about your project requirements.

Simplified Workflows: Final Column Step is Optional

We understand that protein labeling kits are readily available from a number of sources, and the differences between these kits may not be immediately apparent. With names like EasyLink (Abcam) and Lighting-Link™ (Innova Biosciences), one-step labeling kits from other companies promise quick protein labeling and maximum yields—up to 100%. However, both of these features stem from the fact that these kits do not include a final column purification step to remove free dye, as do several Molecular Probes® protein labeling kits. To meet this challenge, we decided to test several of our own protein labeling kits with a stripped-down workflow to see if the final column purification step was absolutely necessary to produce labeled protein conjugates suitable for immunocytochemical staining.

We prepared labeled secondary antibodies using three of our most popular protein labeling kits—the Alexa Fluor® Protein Labeling Kit, Alexa Fluor® Monoclonal Antibody Labeling Kit, and SAIVI™ Alexa Fluor® Antibody Labeling Kit. For each kit, we followed either: 1) the standard protocol including the final column purification step to remove free dye, or 2) a simplified protocol in which the final purification step was omitted.

As shown in Figures 1 and 2, all three Molecular Probes® protein labeling kits produced fluorescent conjugates that effectively stained cells, even without the column purification step. As expected, the standard protocol with column purification produced slightly higher signal:noise ratios (Figure 2); however, we encourage researchers to consider whether the column purification step would significantly alter the outcome of their experiments. Furthermore, we found that the addition of Image-iT® FX signal enhancer (available separately) to cells prior to staining with the labeled protein conjugate reduced the slight background fluorescence due to the presence of free dye, producing results that were nearly indistinguishable from those obtained with a column-purified conjugate (Figure 2A).

More importantly, we found that, even without the column purification step, the Molecular Probes® protein labeling kits produced fluorescent conjugates were far superior to those of the other one-step labeling kits tested, in terms of signal strength and background fluorescence (Figures 2 and 3). Thus, with this new simplified workflow, Molecular Probes® protein labeling kits provide one-step labeling convenience with high yields and bright results. We offer a wide selection of protein labeling kits for covalently labeling 20 μg to 3 mg protein with a range of Alexa Fluor® dyes as well as biotin and several classic fluorophores, including fluorescein, Oregon Green® 488, and Texas Red® dyes (Table 1).

Figure 1. Comparison of immunofluorescent staining with protein conjugates prepared using Molecular Probes® labeling kits with simplified workflows. Mouse anti–human IgG labeling of HEp-2 cells on prefixed test slides (INOVA Diagnostics, Inc.) was detected with fluorescent goat anti–mouse IgG secondary antibodies prepared using the indicated kits, with or without the optional column purification step (and in one image, with Image-iT® FX signal enhancer). All images were acquired under identical capture conditions (i.e., 20x objective, 150 msec exposure time and Semrock® BrightLine Alexa Fluor® 488 filter set for Alexa Fluor® 488 conjugates, 500 msec exposure time and Semrock® Alexa Fluor® 647 filter set for the Alexa Fluor® 647 conjugates) using a Zeiss® Axioskop2® fluorescence microscope.

Figure 2. Signal:noise ratios for immunofluorescent staining with protein conjugates prepared using different labeling kits. Mouse anti–human IgG labeling of HEp-2 cells on prefixed test slides (INOVA Diagnostics, Inc.) was detected with (left) green-fluorescent or (right) far-red–fluorescent goat anti–mouse IgG secondary antibodies prepared using the indicated labeling kits and protocols. The image capture conditions were identical to those used in Figure 1, and the signal:noise ratios were quantitated using SlideBook™ 5 software.

Figure 3. Immunofluorescent staining with protein conjugates prepared using three different protein labeling kits. Microtubules of bovine pulmonary aorta endothelial cells (BPAECs) were tagged with mouse monoclonal 236-10501 anti–bovine α-tubulin antibody and then probed with goat anti–mouse IgG secondary antibodies that were conjugated with fluorophores using the following protein labeling kits: (A) EasyLink Cy5® Conjugation Kit (Abcam) and accompanying protocol; (B) Lightning-Link™ FluoProbes® 647H (Innova Biosciences) and accompanying protocol; or (C) SAIVI™ Alexa Fluor® 647 Antibody Labeling Kit and one-step labeling protocol without column purification. All images were acquired under identical capture conditions (i.e., 40x objective, 500 msec exposure time, Semrock® Alexa Fluor® 647 filter set) using a Zeiss® Axioskop2® fluorescence microscope.

Effective Antibody Labeling Even When Other Proteins Are Present

When working with crude antibody preparations in serum, ascites fluid, or hybridoma supernatants, simple one-step labeling kits won’t work without first purifying the antibody. As an alternative, we offer the APEX® Antibody Labeling Kits for labeling 10–20 μg of IgG even in the presence of proteins or other contaminants. The APEX® kits utilize a solid-phase labeling technique that captures IgG on resin in the provided APEX® antibody labeling pipette tip. Once contaminants are washed through the tip, the captured IgG is labeled with a reactive fluorophore and then eluted from the resin. In as little as 2.5 hours (with minimal hands-on time), the fluorescent IgG is ready for use in a fluorescence imaging or flow cytometry experiment.

The Zenon® Antibody Labeling Kits also provide rapid and quantitative labeling of 1–20 μg of IgG, starting with crude antibody preparations in serum, ascites fluid, or hybridoma supernatants. This noncovalent labeling method takes advantage of the immunoselectivity of antibody binding by forming a complex between an IgG and a fluorophore-labeled Fab fragment directed against the Fc portion of the IgG. The Fab–IgG complex is formed in under 10 minutes, with no pre- or post-labeling required, and is immediately ready to be used to stain cells and tissues.

Table 1. Molecular Probes® Protein Labeling Kits.

For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.