MICA and its receptor NKG2D were expressed as an inclusion bodies in E coli. It was therefore denatured and renatured employing dialysis method of refolding. Carrying out ELISA of MICA and it antibody was positive. We didn't have antibody of NKG2D but because the it has a HIS-tag, we performed the ELISA of NKG2D with anti-HIS-tag and the result was positive. We tried to use ELISA to test the binding affinity of these to proteins ( i first coated NKG2D, then MICA, then the antibody of MICA) but the results was negative. I would like to know if i did the right thing by using ELISA to test their affinity and if so what could be the reason why the results was negative and is there a way i can improve on the ELISA to get positive results. Also i would be grateful if you can suggest alternative method i can use to test their affinity. Thank you.
I feel there could be two possible reasons for the failure. first, the protein that you recognised in ELISA using anti-HIS tag was not the desired protein(could have been some unwanted protein with a long HIS strech) or , possibly, denaturation -renaturation steps deformed the native structure of binding doamin of either or both proteins. In this situation afinity electrophoresis could be used to start with. later on, more advanced techniques ( BiFC, fluoroscent anisotropy or calorimetry )could be used to confirm results.
Thank you very much Dr. Sagar for your insightful suggestions, I will try what you have suggested.
You might want to employ MicroScale Thermophoresis (MST) to investigate your interaction!
In addition to the binding affinity, the stoichiometry and other parameters of an interaction can be studied applying MST. Basically, you label one molecule with a fluorescent dye (or you express your protein as a fusion protein); for the ligand you prepare a dilution series which is added to constant amounts of the fluorescent protein. You only need little amounts of your sample (4µl per titration point) with nM concentrations of the fluorescent protein.
So it is very easy to set up an MST experiment, the instrument is straightforward to use and the measurement is really fast (it takes approx. 10min to obtain a Kd using 16 titration points)!
For further information, you could have a look at our homepage:
http://www.nanotemper-technologies.com//home/
And please feel free to contact me via [email protected]!
Best regards,
Heidi
Thank you very much Dr. Roth. I really appreciate your help. I will read around this method and if a have any question or difficulty, i will contact you. Thank you.
Hi Desmond, binding characterization with SPR is of course an option to extract kinetics and affinity of protein interactions. But since you are working with cells and cellular receptors there is an alternative technique, LigandTracer, that extracts the same binding information as with SPR but measures interactions of ligands that bind to receptors on living cells. This technique uses labels and by labeling individual components like the anti MICA antibody you can follow the MICA binding.
Besides the answers you already got, the ELISA could have simply failed because the NKG2D/MICA interaction is very dynamic (quick binding and dissociation) so that the complexes dissociate in ELISA during washing before you actually got to the quantification step.
Best regards,
Jos
Thank you very much Dr. Job for your suggestions, i will try them and get in touch later.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology