TrailBlazer Tag and TrailBlazer StarBright Dye Label Kit - Troubleshooting

Leaking of the Filter in the TrailBlazer Tag Kit during step 1

The volume of the antibody and Conjugation Buffer in column exceeds 500 µl due to air bubbles present, resulting in leaking when cap is closed

1. Avoid introducing air bubbles when adding the antibody and Conjugation Buffer to the Filter.

2. Provided the antibody is added before the Conjugation Buffer any leaking will be minimal and will not significantly affect the conjugation process.

No signal

1. The primary antibody does not work for the application.

1. Verify your antibody works in your application with a secondary antibody prior to labeling.

2. The purified antibody concentration is unsuitable for conjugation.

2. The recommend antibody concentration is 0.2 mg/ml or greater, antibodies lower than this will need to be concentrated prior to labeling. Note: concentrating the antibody  may also concentrate large proteins such as BSA.

3. Antibody buffers or additives in the purified antibody are unsuitable for conjugation.

3. Verify that the antibody buffers and additives are compatible with the kit.

4. The antibody with a SpyTag is degraded.

4. Check that the SpyTagged antibody has been stored at 4^oC and was used within two weeks.

Poor performance of StarBright Dye conjugated antibody, with low stain index in flow or low limit of detection (LOD) in western.

1. Low antibody yield / concentration of the StarBright Dye labeled antibody.

1.1. The antibody will be too dilute for optimal labeling if the antibody was eluted into greater than 100 µl of Conjugation Buffer during the antibody preparation step of the TrailBlazer Tag Kit.

1.2. Ensure the recommended centrifugation force was used to prevent loss of antibody, especially when using desalting columns in the TrailBlazer Tag Kit.

1.3. Ensure the correct number of washes for each wash step was performed.

2. Too much BSA carryover after antibody preparation step of the TrailBlazer Tag Kit.

2. Check BSA tolerance guidelines in the instructions for use have not been exceeded.

3. Presence of unlabeled antibody.

3. If there is improper mixing of the Spin Column during the TrailBlazer Label Kit protocol, unlabeled antibody will not be removed. Ensure an end-over-end rotator was used and the slurry in the Spin Column had not settled during mixing.

4. Low ratio of antibody to dye.

4. If an expired TrailBlazer StarBright Dye Label Kit is used the Spin Column will have reduced activity. The antibody can still be used for downstream applications, but a higher volume of antibody per assay will be required.

Poor stability of labeled antibody.

Incorrect storage.

It is recommended that the StarBright Dye labeled antibody is stored at 4^oC in the dark. If the antibody is diluted, the stabilizers present in the final formulation will also be diluted and could cause instability of the antibody.

High non-specific binding.

1. Too much labeled antibody is used for staining.

1. It is recommended to perform a titration using the same experimental protocol and cell type as in your final assay to determine the antibodys optimal working concentration.

2. Background staining due to the non-specific binding of the original purified antibody, poor quality antibody can lead to poor staining and high background.

2. To check the quality of your purified antibody, run the assay again following an indirect staining protocol, using a labeled secondary antibody. If non-specific staining remains the issue is likely to be a result of a poor-quality purified antibody.

3. Antibody binding to Fc receptors.

3. Including a Fc blocking step before staining will reduce antibody binding to Fc receptors, reducing background staining.

Bright aggregates present.

Antibody labeled to aggregated dye.

Check that the TrailBlazer Label Kit has not expired. A brief centrifugation (5 min at 6,000g) of the antibody prior to staining can reduce the presence of antibody aggregates.

Signal too bright.

Antibody used at suboptimal concentration in the flow assay. For example, if an antibody binds to a high antigen density marker, there will be a large separation of the positive and negative population, this could be off scale if the antibody concentration is not optimal.

A titration using the same experimental protocol and cell type as in your final assay should be performed to determine the antibodies optimal working concentration.

High background.

Background staining due to a sub-optimal experimental protocol or the suitability of the primary antibody for the application or protocol.

1. Check that the primary antibody is suitable for the western blot and the sample tested.

2. The antibody was not used at its optimal dilution. A titration using the same experimental protocol as in your final assay should be performed to determine the antibody’s optimal working concentration.

3. There could be many reasons for high background, that are independent of the antibody. Here is a detailed list of reasons for background issues and potential solutions.