10 most popular questions from the Immuno-PCR webinar

Here are the 10 most popular questions from the Immuno-PCR webinar

1. How can I confirm that my antibody-oligo conjugation has been successful?

The best way of doing this is to use the conjugate in your downstream application, however it’s also possible to analyze your antibody-oligo conjugate by gel electrophoresis. By running a small amount of your conjugate on a reducing SDS/PAGE gel, and staining for protein, you should be able to see the antibody heavy and light chains, which will demonstrate a band shift when they are bound to the oligo. The size of the band shift will be dependent on the size of the oligo.

2. Once the antibody and the oligo have been activated, you say that they require desalting. Where can I purchase desalting columns, and how do they work?

The Thunder-Link® PLUS oligo conjugation kit is provided with all the reagents that you require to carry out your antibody-oligo conjugation, including the desalting columns. These columns work by gravity flow, and are packed with sepharose beads which contain pores. Larger molecules such as antibodies or oligos will pass through the column relatively quickly since they will not become trapped by the pores, and they will therefore exit the column before any smaller molecules such as salts.

3. If my antibody is not at a concentration of 1mg/ml, can I still conjugate it using Thunder-Link® PLUS?

You certainly can. If your antibody is in a buffer which is compatible with Thunder-Link® PLUS, and is at a concentration of greater than 1mg/ml, then you can simply dilute it to 1mg/ml using the wash buffer that’s provided in the Thunder-Link® PLUS kit. If your antibody is at a concentration of less than 1mg/ml, you can use the appropriate antibody purification kit from our AbSelect™ range to quickly and easy concentrate your antibody, or to perform a simple buffer exchange.

4. Can I use the Thunder-Link® PLUS kit to conjugate an IgM antibody?

Although our Thunder-Link® PLUS conjugation protocol was optimized for IgG antibodies, the kit is compatible with other antibody subtypes such as IgM. Please get in touch with our technical support team for advice.

Please can you explain again the difference between direct and indirect immunodetection?

Direct detection relies on the use of an antibody which has been directly labeled with a detection moiety such as an enzyme or a fluorescent dye, while indirect detection requires the use of a labeled secondary antibody. Directly-labeled antibodies are often preferred, since by using these the assay will require fewer incubation and wash steps, and the background staining which may be introduced through the use of secondary antibodies can be avoided.

6. Are you able to supply me with oligos?

Yes we can, although this will be subject to a small admin fee.

7. Can I use Thunder-Link® PLUS to conjugate an antibody to RNA?

Unfortunately, the reagents that are provided in the Thunder-Link® PLUS conjugation kit are not RNase free, so the kit is unsuitable for conjugating an antibody to RNA. We can however perform this service for you as custom work – please get in touch to discuss this further.

8. How much more sensitive is immuno-PCR than an ELISA?

While the limit of detection of an ELISA is within the µg-pg range, immuno-PCR offers detection in the pg-fg range. Although the limit of detection of an immuno-PCR assay will vary depending on the choice of antibody, immuno-PCR can often detect the antigen at a 1000-fold lower concentration than that which would be detected by an ELISA developed against the same target.

9. Can I use my antibody-oligo conjugate for a technique other than immuno-PCR?

Definitely. Antibody-oligo conjugates are ideally suited to immuno-PCR, but they’re also regularly used for assays such as proximity ligation or proximity extension. Both of these techniques rely on the use of two oligo-conjugated antibodies against different epitopes of the same protein, or against two biomolecules which have the potential to interact with one another. When both antibodies are bound to their targets and are therefore close to one another, the two oligos will hybridize and the DNA can then be amplified and detected.

10. What would you suggest to be the main advantages of immuno-PCR?

Immuno-PCR combines the specificity of an ELISA with the signal amplification of PCR. As well as being specific and highly sensitive, it offers the opportunity for multiplexing, allowing several readouts to be obtained from the same precious sample material.

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