深圳欣博盛生物科技有限公司

1. Do I have to use both TrailBlazer Tag and StarBright Dye Label Kits to label a StarBright Dye to my antibody?

· 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

2. What is the shelf life of the TrailBlazer Tag and StarBright Dye Label Kits?

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

3. What are the TrailBlazer Tag and StarBright Dye Label Kits pack sizes?

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

4. What are the shipping and storage conditions for TrailBlazer Tag and StarBright Dye Label Kits?

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

5. Can the TrailBlazer StarBright Dye Label Kit be used to label proteins other than antibodies?

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

6. How do I select which TrailBlazer StarBright Dye Label Kit to use?

· 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

7. What chemistry does the kit use?

· 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

8. Is there a specific purified antibody concentration that is optimal?

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

9. Is it necessary for the purified antibody to be in a buffer with a specific pH?

· 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

10. How much of the final StarBright Dye labeled antibody should I use in downstream applications?

· 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

11. Can the antibody preparation step be omitted from the TrailBlazer Tag Kit if using an antibody in PBS + 0.09% sodium azide?  

· 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

12. Will any uncoupled antibody be present in the final product?

· No, any unlabeled antibody is removed at the end of the TrailBlazer StarBright Dye Label Kit protocol

13. What is the expected antibody yield/recovery?

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

14. What is the shelf life of the StarBright Dye TrailBlazer Kit labeled antibody?

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

15. What is the shelf life of the StarBright Dye labeled antibody, conjugated by the TrailBlazer StarBright Dye Label Kit?

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

16. Can I use more than one StarBright Dye TrailBlazer Kit labeled antibody in multiplexing experiments?

· 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

17. What are the benefits of using TrailBlazer StarBright Dye Kits over LYNX or ReadiLink Kits?

· 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

18. Why should I directly label my antibodies using TrailBlazer Kits rather than using biotin-streptavidin or primary-secondary antibody staining protocols?

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

o 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

o No additional controls are required, such as secondary alone

o 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

o 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

o 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

Additional information (non-customer facing)

1. Can we tell customers the mechanism of the SpyTag binding to antibody?

· Yes, as it's not proprietary chemistry. A customer can be told the Tag Kit works by thiolating the antibody and reacting it with a maleimide derivative of the SpyTag

2. Can I use a recombinant full length SpyTag antibody with the Label Kit?

· Yes, but the exact protocol has yet to be optimized. From R & D: I don't think we should be making recommendations for using recombinant antibodies yet, when we only have experience using one and even that is limited. That said, here is how I would approach it. For spy-Ab made with kit 1, the mass ratio of catcher-Pdots to Ab is roughly 3:1. If we want the same ratio for recombinant spy-Ab then we should recommend the customer add 70 ug of Ab to the Label tube in kit 2. The concentration of the Ab should be at least 1 mg/ml. Higher concentrations is ok but if the concentration is too low then the reaction volume won't fit in the ACHT spin column.

3. Can the kits be used to label a Fab recombinant antibody?

· This would be antibody dependent, and a customer would have to try, some adjustments may be needed.

· The R & D team have tried using conventional StarBright Dye chemistry and found different kinds of recombinant Fab's worked better than others, which might be related to size. They were able to compensate for less-than-ideal conjugation by adjusting the ratio of Fab to nanoparticle in the conjugation reaction. This could be the case for labeling kits, but without testing it’s not clear.

4. Is the labeling site specific?

· No. Our tagging chemistry is not regarded as site-specific. The first step, treatment with iminothiolane, reacts with lysines. The IgG has dozens of these, and only a few react. It is thought that how lysines react is more or less random, hence the lack of site specificity.

5. Given their mechanism, are these kits compatible with antibodies in buffers containing amines? If so, how can the amines be blocked to avoid non-specific binding?

· The first step, with the filter in the Tag Kit, serves to exchange the antibody into a compatible buffer. In theory, the presence of amines in the antibody formulation shouldn't matter, as there won't be any amines around to "block" or otherwise deal with

6. What range of antibody amount can I label?

· It has been developed to label 100 µg antibody. Other amounts have not been tested. Using a little less should be fine, but the conjugation efficiency will start to drop, so we would only recommend that customers use 100 µg.

7. Can I use the labeled antibody to stain fixed cells in flow cytometry?

· It will depend on the clone. Crosslinking during the fixation process can disrupt the epitope so the antibody may no longer recognise it. This will be antibody clone dependent. If the purified version can be used to stained fixed cells, then the Kit labeled antibody will also be suitable

8. Can cells stained with the Kit labeled antibodies be fixed in alcohol?

· No, we recommended using an alternative fixative. Internal testing has shown that the staining is reduced if cells are fixed in alcohol (MeOH and EtOH) post staining

9. Can cells stained with the Kit labeled antibodies be fixed in 4% PFA?

· The brightness of the signal can be reduced, so we recommend only using up to 2% PFA. Internal testing showed 1 and 2% did not affect staining, but increasing to 4% the brightness and/or population identification was slightly affected

10. Will the Label Kit still work if it was accidently frozen, for example during shipping?

· The StarBright Dye will be damaged by freezing, therefore we would not recommend a customer uses the Kit

11. Will the Tag Kit still work if it was accidently frozen, for example during shipping?

· Both the Filter and the desalting columns can be damaged by freezing, therefore we would not recommend a customer uses the Kit

12. Could the SpyTag bind to the antibody binding site and prevent it binding to its epitope?

· The site of SpyTag attachment can be anywhere on the antibody. As it is random, only a small percentage of any antibody would have its binding site modified. We have never had an issue that can be attributed to this effect

13. In flow cytometry customers can check their antibody has been labeled by staining comp beads. Is there a simple way using western blotting to confirm successful antibody labeling?

· Yes, for mouse antibodies a customer can use their kit labeled antibody to blot protein G. Any mouse antibody will bind to the blotted protein G through the Fc region. This method can be used to determine whether the antibody product is successfully labeled

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