Could anyone give me some suggestions about the "45Ca2+ blot overlay assay "?
I searched for the procedure but there are some points that I could not understand.
The general protocol is described as below:
1. Run the protein on the SDS/PAGE
2. Transfer onto PVDF membrane
3. Remove the membrane from the blotting chamber, wash briefly with blotting buffer
4. Wash the membrane in washing buffer and shake for 20 mins.
5. Place membrane in a plastic bag, add probing buffer which contains 45Ca2+, incubate 10 mins with gentle agitation.
6. Transfer membrane onto plastic container and add aqueous ethanol, shake in 5 mins
7. Remove the membrane from the container, air dry for at least 4 hrs in room temperature
8. Expose the dried membrane for 1 hr- 3 days at room temperature.
So, is it that we don't need to incubate the protein with antibody anymore? If so, how could we determine the protein of interest directly interact with Calcium?
If possible, could anyone who has done this experiment give me the detail protocol?
Thank you so much for your help.
45Ca+2 is a radionuclide. If a protein in the PVDF membane binds calcium it will appear in the autoradiography. To corroborate that your protein interacts with calcium you must perform a western blot. If the signal of the calcium autoradiography correspond with the one in the western blot you could say, as a first aproach, that they interact.
Maybe protein takes a different conformation when it binds to Ca2+ ion. Then your antibody is specific to the conjugate of Ca and protein.
45Ca+2 is a radionuclide. If a protein in the PVDF membane binds calcium it will appear in the autoradiography. To corroborate that your protein interacts with calcium you must perform a western blot. If the signal of the calcium autoradiography correspond with the one in the western blot you could say, as a first aproach, that they interact.
Thank you so much for your suggestions, they are really helpful for me and I do appreciate them.
Now I am no more confused about the procedure. So, I will perform the SDS-PAGE and membrane transfering, then incubate with 45Ca+2 followed by Calcium autoradiography. Is that right?
Hopefully I could get the result.
Thank you so much for all your help.
Regards
H.T.Xoan
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