How to determinate concentration of conjugated Ab`s ? - (Sep/18/2012 )

Dear All
I have conjugated two Ab`s in the way that it is possible to have:
- one Ab1 and three Ab2 toghter
- one Ab1 and two Ab2
- one Ab1 and one Ab
and my question is - how I can determinate protein concentration of such conjugates?

I guess first combination will gives more fluorescence (+++), second less (++), for third once you are not using Ab2 or what? will get differences in intensity.
you have to use each for different experiments with brighter colors but with same ultimate resulting curve/result.

MicroBone on Tue Sep 18 13:44:44 2012 said:

This question is interesting one. Today when talking to friend I found that this i\type of conjugation is done to study antigen interaction study. where secondary antibody will bind to two or more antigen based on how many antigen interaction u are studying. my previous explanation may need modification if its antigen interaction study.

using the common protein estimation methods should work

I'm guessing you want protein concentration of each possibility. You might want to separate by gel filtration. Ab1 and Ab2 are going to have about the same MW if they are both IgG; that doesn't vary by much. But two Ab conjugated together will have twice the molecular weight, a conjugate with one Ab2 and two Ab2 is going to have 3 times the molecular weight of IgG and so on. Given the size differences you should be able to separate based on size and go from there. You can get a ug/mL value from a Bradford assay or A280 but the number of moles is going to depend on the number of Ab you have stuck together.