In the literature (Anal. Chem. 1995, 67, 3526-3535), the bio-interaction between the hapten with the same moieties and lgG antibody from another species has been studied. The first Kd is about 1.9±0.4 µM and the second Kd is about 8.5±0.9 µM when the hapten is single charged. In the microfluidic device surface, the first Kd is 24.6 µM. So in the aqueous phase, the first Kd is smaller than the one immobilized on the surface. This is because the hapten and protein are free in the solution and no extra entropic cost for bringing the protein to the surface and proper orientation for binding.
I am not sure that this technique could be used to study bio-interaction in the free solution. If it could and we separate the free protein and the binding complex and measure the fluorescence of the complex, I think the equation 10 will not change and the shape of Figure 7 will not change. I don't think that I answered the question:). It seems I don't understand the question well.
Clarification for question 2.
Figure 7 shows KD(app) versus available ligand density. If one were to do a different experiments, using a different technique, in which the ligand is free, what changes will be observed in the plot? Use the answer in question 1 to come up with an answer for question 2.
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Comments
In the system described in the paper, the antibody is bivalent and binds to two haptens immobilized onto an attached phospholipid bilayer.
1. What would be the expected Kd's if the antigens were free in solution?
2. How would Figure 7 and Equation 10 change when the haptens are free in solution?
Posted by: Edgar Arriaga | April 23, 2008 12:31 AM
In the literature (Anal. Chem. 1995, 67, 3526-3535), the bio-interaction between the hapten with the same moieties and lgG antibody from another species has been studied. The first Kd is about 1.9±0.4 µM and the second Kd is about 8.5±0.9 µM when the hapten is single charged. In the microfluidic device surface, the first Kd is 24.6 µM. So in the aqueous phase, the first Kd is smaller than the one immobilized on the surface. This is because the hapten and protein are free in the solution and no extra entropic cost for bringing the protein to the surface and proper orientation for binding.
I am not sure that this technique could be used to study bio-interaction in the free solution. If it could and we separate the free protein and the binding complex and measure the fluorescence of the complex, I think the equation 10 will not change and the shape of Figure 7 will not change. I don't think that I answered the question:). It seems I don't understand the question well.
Posted by: Jing Zhang | April 23, 2008 11:40 PM
Clarification for question 2.
Figure 7 shows KD(app) versus available ligand density. If one were to do a different experiments, using a different technique, in which the ligand is free, what changes will be observed in the plot? Use the answer in question 1 to come up with an answer for question 2.
Posted by: Edgar Arriaga | April 26, 2008 09:27 AM