TrailBlazer Tag and TrailBlazer StarBright Dye Label Kits

Tag it - Label it - Use it - Store it

TrailBlazer™ Tag and StarBright™ Dye Label Kits TrailBlazer™ Tag and StarBright™ Dye Label Kits allow you to label your antibody to superior StarBright Dyes using innovative SpyTag and SpyCatcher technology. They provide the benefits of using StarBright Dyes in your flow cytometry assay and western blot for your antibody of choice.

  • Broad antibody compatibility: label any purified antibody greater than 0.2 mg/ml in a range of buffers and isotypes
     
  • High performance: comparable to catalog labeled antibodies
     
  • Long term stability: labeled antibody is stable for a year
     
  • Easy to follow protocol: Simple protocol with minimal hands-on time
     
  • Superior dyes: all the benefits of StarBright Dyes on your antibody of choice in any panel:
    • Flow cytometry: bright, with narrow excitation and emission profiles
    • Western blot: bright, highly sensitive fluorescent detection with short exposure times
       
  • Large label choice, expanding multiplexing options:
    • Flow cytometry label kits for StarBright Blue 700, StarBright Violet 440, StarBright Violet 515, StarBright Violet 570, StarBright Violet 610, StarBright Violet 670 available now. Watch this space as more coming in 2025 covering all 32 StarBright Dyes
    • Western blot label kits for StarBright Blue 700. StarBright Blue 520 coming in 2025

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Innovative Technology and Protocol


TrailBlazer Tag and Label Kits allow purified antibodies to be labeled with a wide range of StarBright Dyes, by utilizing a simple 2-kit process (Figure 1).

Innovative SpyTag and SpyCatcher technology, also termed ‘molecular superglue’ is used to ‘glue’ StarBright Dyes to antibodies (Keeble et al. (2017)). Firstly the SpyTag is attached to your antibody using the Tag Kit (Figure 1. A). This antibody (or a recombinant antibody that already has a SpyTag) is mixed with a StarBright Dye labeled SpyCatcher using the Label Kit (Figure 1. B).

A spontaneous reaction occurs between the SpyTag and SpyCatcher resulting in an irreversible covalent isopeptide bond. This is what makes the TrailBlazer conjugation kits yield highly stable, labeled antibodies. The SpyTag and SpyCatcher technology is also used in our custom HuCAL® recombinant antibody generation service, for TrailBlazer Antibodies. 

 

Fig 1. Overview of TrailBlazer Tag and StarBright Label Kit.


Fig 1. Overview of TrailBlazer Tag and StarBright Label Kit. A. Attachment of a SpyTag to a purified antibody. B. Conjugation of a StarBright Dye Catcher to a SpyTag labeled antibody. 

Simple to Use


The TrailBlazer Kits are simple to use with minimal hands-on time (Table 1). They have been designed to fit with the way you work, enabling other tasks to be performed alongside due to short hands-on time required.

Make use of a handy protocol worksheet to record each step of the conjugation process, including antibody preparation and modification, SpyTag attachment and StarBright Dye labeling.

Download this template to keep track of what you have done.

Table 1. Typical time taken to complete Tag and Label kits 

TrailBlazer Kit

Hands-on time 

Total time 

TrailBlazer Tag Kit 

40 min 

2hr 15 min 

TrailBlazer StarBright Dye Label Kit 

10 min 

1hr 40 min*

 * Longer if using an overnight incubation

As the TrailBlazer Kits have been designed for easy integration into your workflow, they are conveniently compatible with a variety of antibody isotypes (Table 2) and buffers (Table 3). 

Table 2. Isotype Compatibility

 Isotype 

Compatibility

 Mouse IgG2a 

​

 Mouse IgG2b 

​​ ​

 Mouse IgM 

 ​​

 Rat IgG1 

​​

 Rat IgG2a 

​​

 Rat IgG2b 

​​

 Rabbit IgG 

​​

Table 3. Buffer Compatibility

 Buffer 

Compatibility

 PBS 

​​

 0.5% BSA 

​​

 0.09% sodium azide 

​​

 50mM Tris 

 0.01% Thiomersal 

High Performance Conjugated Antibodies


Not only are the StarBright Dye antibodies labeled by TrailBlazer Kits comparable to commercial manufactured labeled antibodies, they also have the advantages of the StarBright Dye. For example, antibodies conjugated to StarBright Violet 440 are significantly brighter than those of a comparable fluorophore conjugated using a Readilink Conjugation Kit (Figure 2). This makes them ideal to detect markers with a low antigen density in flow cytometry or less abundant proteins by western blotting.

Fig. 2. Comparison of antibodies conjugated using TrailBlazer and Readilink Conjugation Kits.


Fig. 2. Comparison of antibodies conjugated using TrailBlazer and Readilink Conjugation Kits.  A. Mouse Anti-Human CD4 (MCA1267) or B. Mouse Anti-Human CD8 (MCA1226) was tagged and labeled using TrailBlazer Tag Kit (12020038) and StarBright Violet 440 Label Kits (12020042) (red) or Readilink 405/454 Antibody Labeling Kit (135011) (blue). Human PBMCs were stained in the presence of 10% human serum. Data acquired on the ZE5 Cell Analyzer.

Benefits of TrailBlazer StarBright Dye Label Kits for Flow Cytometry

Fluorophore choice is no longer a limitation: increase your fluorescent options for multiplexing panels of any size. StarBright Dye label kits covering all five common laser lines will be available, making panel design easier.  

Antibodies conjugated with StarBright Dyes are suitable for use in both surface staining and intracellular staining, as described in our optimized protocols.

Bright with Exceptional Performance


TrailBlazer Kit labeled antibodies are bright with improved or equivalent performance to directly conjugated antibodies.

Fig. 3. Human CD4 antibodies conjugated to StarBright Dyes are as bright as commercially manufactured conjugated antibodies.


Fig. 3. Human CD4 antibodies labeled to StarBright Dyes are as bright as commercially manufactured conjugated antibodies. Mouse Anti-Human CD4 (MCA1267) was tagged and labeled using TrailBlazer Kits (red) and compared to Mouse Anti-Human CD4 directly conjugated StarBright Dye (blue). Human PBMCs were stained in the presence of 10% human serum. Data acquired on the ZE5 Cell Analyzer.

High Stability


TrailBlazer Kit labeled antibodies are highly stable due to the TrailBlazer technology used. SpyTagged antibodies are combined with a StarBright Dye SpyCatcher generating an irreversible bond. This yields a highly stable antibody; when stored at 4oC in the dark it is stable for a year with minimal changes in brightness. This avoids the need to perform multiple label reactions when the antibody is used for longitudinal studies.

Fig. 4. High stability of human CD4 antibody 1 year after conjugation to StarBright Blue 700 using TrailBlazer Tag and Label Kits.


Fig. 4. High stability of human CD4 antibody 1 year after conjugation to StarBright Blue 700 using TrailBlazer Tag and Label Kits. Mouse Anti-Human CD4 (MCA1267) was tagged and labeled using TrailBlazer Kits (12020038 and 12020040). 1 year after storage at 4oC it was compared to a directly conjugated Mouse Anti-Human CD4 StarBright Dye (MCA1267SBB700) (blue). Human PBMCs were stained using 5 µl of antibody in the presence of 10% human serum. Data acquired on the ZE5 Cell Analyzer, n = 3.​

Isotype Compatible


TrailBlazer Kit Tag and Label Kits are suitable to use with common isotypes.

Fig. 5. TrailBlazer Kit isotype compatibility.


Fig. 5. TrailBlazer Kit isotype compatibility. Human PBMCs were stained with antibodies labeled using TrailBlazer Tag and Label Kits (red) and compared to directly conjugated catalogue antibodies (blue). A. Mouse Anti-Human CD4 (RPA-T4 clone) B. Mouse Anti-Human CD4 (OKT4 clone) C. Rat Anti-Human CD195 (HEK/1/85a clone). Data acquired on the ZE5 Cell Analyzer.​

Flexible to Work with Different Purified Antibodies


These TrailBlazer Kits are very versatile as they are compatible with a wide range of buffer types.

Fig. 6. Compatibility of TrailBlazer Tag and StarBright Dye Kits with purified antibodies containing 0.5% BSA.


Fig. 6. Compatibility of TrailBlazer Tag and StarBright Dye Label​ Kits with purified antibodies containing 0.5% BSA. Mouse Anti-Human CD4 (MCA1267) was tagged and labeled using TrailBlazer Kits (12020038 and 12020045) containing no BSA (red) or spiked with BSA to a concentration of 0.5% (green). Kit labeled antibodies were compared to a Mouse Anti-Human CD4 directly conjugated StarBright Dye (MCA1267SBV570) (blue). Human PBMCs were stained in the presence of 10% human serum. Data acquired on the ZE5 Cell Analyzer.

Reproducible Staining


TrailBlazer Kit Tag and StarBright Dye Label Kits give highly reproducible, consistent staining across the different formats.

Fig. 7. TrailBlazer Kit reproducibility.


Fig. 7. TrailBlazer Kit reproducibility. Human PBMCs were stained with Mouse Anti-Human CD4 (MCA1267) labeled to StarBright Dyes using TrailBlazer Tag and Label Kits. Consistent percentage of CD4+ cells were seen across the different Label Kits. Data acquired on the ZE5 Cell Analyzer. Error bars show SD of triplicate staining.​

Compatible with Common Staining Protocols


TrailBlazer Kit labeled antibodies are suitable for use in your current assay, they are compatible with all common staining protocols tested, meaning no adjustments are necessary. They give highly reproducible data when cells are stained and fixed in a range of common buffers and fixatives.

Fig. 8. TrailBlazer Kit compatibility.


Fig. 8. TrailBlazer Kit compatibility. Mouse Anti-Human CD4 (MCA1267) was labeled to StarBright Violet 440 using TrailBlazer Tag (12020038) and TrailBlazer SBV440 Label Kit (12020042). Human PBMCs were stained in A) different staining buffers or B) fixed with different fixatives. Data acquired on the ZE5 Cell Analyzer.​

Ideal for Multiplexing


TrailBlazer Kit labeled antibodies are ideal for multiplexing in flow cytometry, multiple labeled antibodies can be used together in standard buffers with no dye interactions.

Fig. 9. Human immunophenotyping panel containing multiple TrailBlazer Kit labeled​ antibodies.


Fig. 9. Human immunophenotyping panel containing multiple TrailBlazer Kit labeled​ antibodies. Human PBMCs were stained with a panel containing TrailBlazer Kit labeled antibodies, directly conjugated antibodies and a viability dye in PBS + 1% BSA. Data acquired on the ZE5 Cell Analyzer.

Suitable for Premixing


TrailBlazer Kit labeled antibodies are suitable for including in mastermixes and premixes with no dye interactions. Ideal when staining multiple samples with the same panel, in a longitudinal study, saving time and improving reproducibility.

Fig. 10. Highly reproducible data of a fresh compared and 1-week premixed cocktail panel containing multiple TrailBlazer Kit labeled​ antibodies.


Fig. 10. Highly reproducible data of a fresh compared and 1-week premixed cocktail panel containing multiple TrailBlazer Kit labeled​ antibodies. Human PBMCs were stained with a panel containing TrailBlazer Kit labeled antibodies, directly conjugated antibodies and a viability dye. The premixed cocktail was stored for 1 week at 4oC prior to staining. Cells were gated on live, single cells (not shown). Data acquired on the ZE5 Cell Analyzer.​

Versatile


TrailBlazer Kit StarBright Dye labeled antibodies are not only suitable to detect cell surface markers, but also intracellular targets too.

Fig. 11. Intracellular Staining using a TrailBlazer Kit labeled antibody.


Fig. 11. Intracellular Staining using a TrailBlazer Kit labeled antibody. Mouse Anti-Human Granzyme B (MCA2120) was labeled to StarBright Violet 440 using TrailBlazer Kits (12020038 and 12020042) and compared to Pacific Blue conjugated Mouse Anti-Human Granzyme B of the same clone. Human PBMCs were stained using Foxp3 Fix/Perm Buffer set (Biolegend, 421403). BD Horizon Brilliant Stain Buffer (BD Biosciences, 563794) was also present during staining using the TrailBlazer Kit labeled antibody. Open histograms show unstained controls and solid histograms show staining with Anti-Granzyme B. Data acquired on the ZE5 Cell Analyzer. 

Benefits of TrailBlazer StarBright Dye Label Kits for Western Blotting 

Bio-Rad’s TrailBlazer Tag Kits (12020038 and 12020901) and StarBright Blue 700 Label Kits (12020040 and 17010362) will label both primary and secondary antibodies for use in western blotting.  

High Sensitivity

TrailBlazer Kit labeled antibodies have performance equal to directly conjugated antibodies, with high sensitivity detecting low amounts of protein with low background staining.

Fig. 12. TrailBlazer Kit tagged and labeled secondary antibody compared with directly conjugated secondary antibody, show equal performance.


Fig. 12. TrailBlazer Kit tagged and labeled secondary antibody compared with directly conjugated secondary antibody, show equal performance. Western blot analysis of Hela lysate spiked with decreasing amount of maltose binding protein (MBP), starting at 20 ng with a 2x serial dilution. Probed with Mouse Anti-Human MBP. Detected with A) Goat Anti-Mouse IgG labeled to StarBright Blue 700 using TrailBlazer Tag (12020038) and StarBright Blue 700 Label Kit (12020040) at 1:1300 or B) Goat Anti-Mouse IgG directly labeled to StarBright Blue 700 at 1:2500 (12004158). Visualized on Chemidoc MP imaging system.

Multiplexing Compatible


TrailBlazer Kit labeled antibodies are idea for multiplexing in western blotting with low spillover into other filters.

Fig. 13. Multiple TrailBlazer Kit labeled antibodies.


Fig. 13. Multiple TrailBlazer Kit labeled antibodies. Western blot analysis of Hela lysate spiked with decreasing amount of maltose binding protein (MBP). Probed with Mouse Anti-Human MBP labeled to StarBright Blue 700 using TrailBlazer Tag (12020038) and StarBright Blue 700 Label Kit (12020040) and Rabbit anti Human GAPDH (VPA00187) detected with Goat Anti-Rabbit IgG StarBright Blue 520 (12005867). Visualized on Chemidoc MP imaging system. Other filters show no spillover.

Isotype Compatible


TrailBlazer Kit Tag and Label Kits are suitable to use with common isotypes.

Fig. 14. TrailBlazer Kit isotype compatibility.


Fig. 14. TrailBlazer Kit isotype compatibility. Western blot analysis of Hela lysate spiked with decreasing amount of maltose binding protein (MBP). A, Probed with Mouse Anti-Human MBP labeled to StarBright Blue 700 using TrailBlazer Tag Kits (12020038 and 12020040). B, Probed with Mouse Anti-Human MBP detected with Goat Anti-Mouse labeled to StarBright Blue 700 using TrailBlazer Kits (12020038 and 12020040). Visualized on Chemidoc MP imaging system.

Super Stable Secondary Antibodies


The technology used in the kit generates stable secondary antibodies for use in western blotting that can be stored for a year at 4oC in the dark with no loss of signal. Therefore, one label reaction can be used in longitudinal studies for a year, saving time and money.

Fig. 15. High stability of TrailBlazer Kit labeled secondary antibodies after 1 year storage at 4oC.


Fig. 15. High stability of TrailBlazer Kit labeled secondary antibodies after 1 year storage at 4oC. Western blot analysis of Hela lysate spiked with maltose binding protein (MBP). Probed with Mouse Anti-Human MBP detected with Goat Anti-Mouse IgG labeled to StarBright Blue 700 using TrailBlazer Tag (12020038) and StarBright Blue 700 Label Kit (12020040). Secondary antibodies were stored at 25oC and compared to a control. The equivalent time in months (shown) for storage at 4oC was calculated based on 15kcal/mol. Visualized on Chemidoc MP imaging system. ​

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Useful TrailBlazer Tag and TrailBlazer StarBright Dye Label Kit Information

StarBright Dyes - Inquiry Form / StarBright Dyes Conjugated Antibodies – Inquiry Form / StarBright Dyes Collaboration Form