oYo-Link® Epitope Tag

Multiplexing

Traditional multiplexing techniques require secondary antibodies that are specific for the primary antibodies, depending on the host and subclass. Due to the relatively small number of available antibody subclasses this limits overall multiplexing capacity. oYo-Link Epitope Tag overcomes this limitation by enabling site specific labeling of the heavy chain of any compatible antibody with 3 x tandem repeat epitope tags. This results in up to 6 x copies per antibody which can be recognized and detected by corresponding anti-tag secondary antibodies separately, therefore allowing multiple antibodies, even from the same host species, to be uniquely identified and detected.

In addition to multiplexing, Epitope Tags can be used for a variety of assays including western blotting, flow cytometry, immunoprecipitation and immunohistochemistry.

1. Q: Will oYo-Link work with my antibody?

A: Please check the antibody compatibility table. Note: oYo-Link Epitope Tag for labeling mIgG1 antibody is not currently available.

2. Q: Can oYo-Link be used to label antibodies in the presence of albumin and/or Tris?

A:Yes, since oYo-Link specifically bind to the heavy chain of IgG, it works in the presence of both albumin and Tris. oYo-Link is compatible with all common buffers. See the full buffer compatibility table here.

3. Q: If an oYo-Link product has accidentally been left at room temperature for a week, will it still work?

A: With the exception of oYo-Link MNase and oYo-Link DBCO, all oYo-Link products are shipped as white or clear pellets and are very stable. While we recommend cold storage, they will not exhibit any loss of activity if left at room temperature for a week. oYo-Link MNase and oYo-Link DBCO must be shipped and stored cold (-20°C) immediately upon arrival, otherwise there will be a loss in activity.

4. Q: Does AlphaThera offer conjugation service?

A:Yes. If you are interested in our conjugation service, please contact us at support@alphathera.local. For custom conjugation services, you will need to send us your antibody. The conjugation and purification fee depends on the size and specifications of each order. Purification is not required for most applications.

5. Q: How many oYo-Link labels will be conjugated to each antibody?

A:For most subclasses and species of antibody, oYo-Link will result in the conjugation of 2 labels per antibody (maximum), one label per heavy chain. For example, ~95% of human IgG and Rabbit IgG will have two labels per antibody. However, there are a few antibodies such as mouse IgG2b and goat IgG, where the conjugation is slightly less efficient. In this case, 60 to 80% of the antibody will be labeled and have a mixture of 0, 1 and 2 labels per antibody. See the antibody conjugation efficiency here

6. Q: How do I know if oYo-Link was successfully conjugated to my antibody?

A: Antibody conjugation can be checked on SDS-PAGE gel. An example is present in the supporting data on each product page.

7. Q: Does 365nm UV illumination damage the antibody and its binding affinity?

A: The conjugated and unconjugated antibody binding affinities have been tested via both ELISA and cell binding assays and don’t show any difference.

8. Q: Do I need to remove free oYo-Link after conjugating to antibody?

A: For most immunoassay applications, purification is not necessary. In our standard recommended protocol, the molar ratio of oYo-Link-to-antibody is 5:1. These conditions ensure that the antibody conjugation efficiency reaches its maximum; however there will be a slight excess remaining in your antibody mixture. This is usually removed during immunoassay washing steps. However, if your application requires high antibody-conjugate purity, please contact us for more information on this process and for a detailed protocol.

9. Q: For oYo-Link click-chemistry products, should I perform the click reaction before conjugation?

A: When working with oYo-Link Azide (Catalog #: AT3002) and oYo-Link DBCO (Catalog #: AT3003), it is required to perform the click chemistry coupling reactions with the molecule of interest first, prior to photo-crosslinking to an antibody. This is to avoid UV illumination damage to the click moiety and to avoid diluting oYo-Link prior to the reaction to maximize the reaction efficiency. For oYo-Link Tetrazine (Catalog #: AT3004) and oYo-Link Thiol (Catalog #: AT3001); it is recommended to perform the coupling reaction before photo-crosslinking. This is to avoid diluting oYo-Link prior to the coupling reaction to maximize the reaction efficiency.

10. Q: What is the lowest amount of antibody that can be labeled by oYo-Link reagents?

A: We recommend working with at least 1 µg of antibody; however, it is possible that even lower amounts of antibody can be labeled.

11. Q: Can antibodies that are highly dilute be labeled by oYo-Link reagents?

A: Yes, antibodies can be efficiently labeled even if diluted to concentrations as low as 50 µg/mL.