Kits: General FAQs

I wondered if your lightning link kits could be used to label proteins other than antibodies? We are working with purified protein. Would the amount of protein used be different from the amount of antibody used? Do you have any references?

The kits are primarily designed to conjugate purified antibodies. However, because they work by targeting free amines they can be used to 'label' other molecules. When, using the kits to label other molecules the standard Lightning-Link rules will apply (i.e. the molecule must be purified, at a high concentration and in an amine free buffer), as the kits have been optimized for antibodies. The optimal amount of material to add to each Lightning-Link mix will have to be determined experimentally. I suggest the use of the 3 x 10ug pack. You may add different amounts of material to each vial and then test the resulting conjugates in your application. This way you can test the technology and obtain some degree of optimization with minimal investment of time, expense and material. Also note that the biotins are attached via a large scaffold molecule. While this increase in mass has no effect on traditional antibody applications it could cause a problem in your application.
Finally, the kits do not target every amine. For example, an amine attached to a large aromatic ring or near an aromatic ring tends to be less reactive. The kits will not work with amides.

We have a commercial Ab (humanized monoclonal) that we wish to label with a couple of different colors of fluorophores. The problems are that the Ab is dilute (20 ug/ml IgG), has significant azide (approx. 0.5% - not clear on this point), and is stabilized with 10 mg/ml BSA. All perfectly reasonable, but inconvenient for NHS labeling (which we have done a lot of). I recently learned of your Lightning-Link technology and wonder how it might be useful for this labeling application. Could you comment on this? I can and will try to contact you tomorrow about this. Of course, we need to find a solution for this, oh yesterday, because we need the labeled Ab in hand on Friday. Even if that doesn't work, please comment on this, without regard to a time schedule.

I would be happy to give advice on the Lightning-Link conjugation kits. You can see example data on the effects of both in these technical guides: Guide 1 and Guide 2. Antibodies also need to need to meet certain concentration/volume/amount guidelines to be suitable for Lightning-Link. For instance, if you add a large volume of a very dilute antibody to a Lightning-Link vial, the conjugation will fail. The concentration, etc. requirements can vary slightly depending on the particular kit (label), but as an example, the requirements for the Lightning-Link fluorescent dye kits are shown in this protocol (bottom of page 1). However, you can treat your antibody to make it suitable for Lightning-Link (provided your antibody volume/amount is not too low).To remove the BSA, you will need to carry out an antibody purification, for instance using the appropriate AbSelect kit. I can help you choose the correct AbSelect kit, if you let me know your antibody species and volume. This purification will also remove the azide. You will then need to concentrate your antibody using the Concentration and Clean Up Kit, which utilizes a simple microfuge spin column method.

*Questions apply to Lightning Link kits only.