TrailBlazer Tag and TrailBlazer StarBright Dye Label Kit - Frequently Asked Questions

Yes, it is a two-step process.

Step 1. Add a SpyTag using the TrailBlazer Tag Kit.
Step 2. Add a StarBright Dye using TrailBlazer StarBright Dye Label Kit.

The shelf life of the TrailBlazer Tag Kit and the StarBright Dye Label Kits is at least 12 months.

The TrailBlazer Tag and Label kits are available in 0.1 mg and 3 X 0.1 mg pack sizes.

The kits will be shipped on ice packs in a cooler box, storage is in the fridge 4-8^oC.

The labeling kits are optimized for labeling antibodies. The dye attaches to amine groups using the SpyTag-SpyCatcher technology. Although amines are present on other proteins, successful labeling is not guaranteed.

This is antibody dependent, and we can’t guarantee successful labeling. The ratio of antibody to SpyTag and StarBright-SpyCatcher has been optimised for full-length antibodies. If the standard protocol is not successful altering the amount of antibody may improve labeling.

Firstly, determine which dyes are compatible for your application and detection instrument. The excitation and emission profiles of StarBright Dyes can be found on Bio-Rad’s Spectraviewer. For multiplexing experiments in flow cytometry, panel design best practices should be followed. Choose StarBright Dyes that have low spillover and spreading with the other fluorophores in your experiment and label antibodies that bind to a low antigen density marker with one of the brightest dyes. For flow cytometry and StarBright Dyes information about brightness can be found in our fluorophore and stain index guides.

TrailBlazer technology is used. The Tag Kit is used to covalently attach SpyTag peptides to lysines, which are amines present on antibodies. The Label Kit covalently attaches a SpyCatcher that is already coupled to a StarBright Dye to a SpyTagged antibody. These strong bonds give the high stability of the antibody-StarBright Dye complex.

The TrailBlazer Tag Kit is designed to work with any concentration at or greater than 0.2 mg/ml.

No, as the buffer is exchanged in the first step of the Tag Kit it will be brought to the optimal pH required for the labeling reactions. Therefore, it will not matter what the pH was originally.

It is recommended to perform a titration to determine the optimal amount for each antibody and experimental condition. As a guide, for flow cytometry typically 5 µl is optimal and for western blotting 1:1000 - 1:5000.

Yes, however the antibody would also need to be at a concentration of 2-5 mg/ml. Using a lower concentration would reduce labeling efficiency which may impact the quality of the final labeled antibody.

No, any unlabeled antibody is removed during the TrailBlazer StarBright Dye Label Kit protocol. The slurry in the Spin Column binds and retains unlabeled antibody, ensuring that the elute contains only StarBright Dye-labeled antibodies. Thus, there will be no interference from unlabeled antibodies in downstream applications.

No, all the StarBright Dye in the Label Kit is conjugated to a SpyCatcher. The ratio of SpyTag labeled antibody and SpyCatcher has been optimized, ensuring there is also no free StarBright Dye labeled SpyCatcher present in the final product.

No, any unlabeled SpyCatchers are removed during the production of the SpyCatcher-StarBright Dye conjugate. As a result, every SpyCatcher that binds to a SpyTag-labeled antibody will be coupled to a StarBright Dye.

No, the chemistry used is not considered site-specific. In the Tag Kit SpyTag will bind to lysine residues, of which antibodies have multiple, although only 2-3 will react.

On average, 2-3 SpyTags bind per antibody.

No antibody aggregation has been observed during development or in the final product. Multiple kit labeled antibodies have been used together in a flow multiplexing panel successfully with no aggregates present.

Any free SpyCatcher sites in the final antibody are caped with the quenching reagent in the Label Kit, which inactivates any residual catcher functionality. Therefore, there are no reactive SpyCatchers available to bind to other SpyTag labeled antibodies and cause aggregation.

Typical yields after using both the Tag and Label Kits are 70-80% of the starting antibody amount.

Upon completion of both the Tag and Label Kits, approximately 500 µL of antibody should be present.

The antibody with a tag has a two-week shelf life, when stored at 4^oC in the dark.

The StarBright Dye labeled antibody has a 12-month shelf life, when stored at 4^oC in the dark.

Yes, the SpyTag-SpyCatcher technology used in the kit covalently links the antibody to the dye. This results in a very strong bond with no transfer of dye to other antibodies.

These kits offer unique fluorescence options that are not available with other kits, this makes them perfect for multiplexing panels as it increases dye choice for improved panel design. You also benefit from the all the unique features of the StarBright Dyes, such as brightness and low spillover and spreading. The StarBright Dye labeled antibody is also very stable with an extended storage for up to 12 months.

For advice on choosing an appropriate conjugation kit, please refer to this article.

There are several reasons why using the TrailBlazer StarBright Dye Kits makes experiments quicker and easier, with improved results:

• As the antibody is directly labeled to a StarBright Dye, staining can be performed in a single incubation step, The other methods require a two-step protocol, firstly incubation with a primary antibody followed by incubation with a secondary antibody. Therefore, staining with a fluorescently labeled antibody is significantly faster
• No additional controls are required, such as secondary alone
• When using secondary antibodies for multiplexing, isotype cross reactivity with other antibodies in your assay need to be considered. This isn’t necessary with directly labeled antibodies. Panel design is therefore simpler, as multiple antibodies of the same isotype can be used in multiplexing experiments
• Using multiple biotin-steptavidin or primary-secondary combinations makes multiplexing panel design complicated, requiring additional optimization. There is no limit to how many TrailBlazer StarBright Dye Kit labeled antibodies can be included in any one experiment
• It is best practice to titrate antibodies prior to use to ensure they are used at an optimal concentration. This is simpler for a directly labeled antibody compared to a secondary antibody