I have conjugated a dye to a goat anti-rabbit secondary antibody. I believe that the more dyes that I conjugate to the antibody, it then loses its ability to recognize the primary antibody. I would like to quantify this, but I am unsure of how to do this. Any recommendations would be greatly appreciated.
Hi Cynthia Schreiber this is an interesting question. The affinity of antibodies to their targets can be measured using plasmon resonance-based technologies (such as Biacore or SPRi) some of these approaches also allow the use of crude samples containing cells instead of requiring the purified antigen.
You can find a reference describing affinity measurements for monoclonal and polyclonal antibodies below: Article Probing Cocaine-Antibody Interactions in Buffer and Human Serum
If you do not have access to this equipment, you can reach out to our team and we would be happy to assist you in finding the best solution for your unique project: https://www.proteogenix.science/bioanalytics/antibody-kd-determination/
Dear Cynthia,
In addition to SPR suggested by Ana Reis one can also use native gel-shift. This would't allow for precise affinity determination, but, if successful, would let you chose the the optimal ratio of fluorescence intensity/affinity in one go. If the only goal is to have a good fluorescent AB, this is probably the easiest approach.
Best
Anton
The easiest way to answer your question is to perform dot immunoblot assay, you can immobilize different protein targets on the nitrocellulose membrane and then incubate with the antibody of interest. If you want to get quantitive results you will need to make a concentration curve and determine at which concentration you will start getting the signal from the dot. The smaller the concentration the higher is the affinity. You can immobilize proteins and primary antibody alike. Don't forget to add a dot with some protein that is known not to bind with your antibody of interest for the negative control. You can read the materials and methods in one of our recent articles where we used this method to confirm the affinity of the antibody produced against mouse proteins to the human protein.
If you can determine molar concentrations of your curve (it is easy for most pure proteins, but not always possible with antibodies i.e. polyclonal antibody) Then you can even calculate the affinity constant as described here: https://www.bio-rad-antibodies.com/antigen-antibody-interactions.html
Article Placental growth factor negatively regulates retinal endothe...
- Badges
- Science topic
- Similar topics
- Chemistry
- Biochemistry
- Biochemical Techniques
- Blotting Techniques
- Immunoblotting
- Dyes