Protein A - FC binding...what really happens?

tmorris at uhnres.utoronto.ca tmorris at uhnres.utoronto.ca
Thu Aug 1 11:21:58 EST 2002


Dear Colleagues:
 
I had always assumed that each IgG FC region could only bind one protein A molecule. The FC portion is referred to in literature in the singular sense, even though it derives from portions of two heavy chains, and this seems to be consistent with crystallographic binding models as best as I can understand them (Deisenhofer, 1981, Biochemistry).
 
However, this may not be the case. Below is an excerpt from an email I wrote to Dr. John Orban, an expert in in biological macromolecules at the University of Maryland.
 
Dear Dr. Orban, I'm sorry to bother you with this, however it seems you are pre-eminently qualified to offer insight into my dilemma. When I incubate fluorophore-conjugated protein-A with protein-A coated beads plus non-specific rabbit IgG, the beads always stain with the flurophore. This was a surprise as this was meant to be a negative control. In the non-control scenario human IgG targets pre-attached to the beads would be probed with anti-human IgG antibody conjugated to fluorescent protein-A. A single FC-bonding site for protein-A would seem to preclude any mechanism by which the bead and fluorescent-A could associate. The beads and fluorescent A together, without IgG, do not stain. So it would appear that IgG can link to at least two protein-A molecules. This is very repeatable and occurs with protein A conjugates also. Very frustrating, but what could be happening. 
 
Here is his reply:
 
Hi Terry: 

The Fc is actually a dimer with two heavy chains and two protein A or G molecules bind per Fc. It may be sterically difficult to get two bead-attached protein A molecules to bind to a single Fc and this may explain why you see fluorescent A binding. Hope this helps. 
John 
 
I was surprised, but do not doubt the integrity of John's assessment. I would like to thow this one open to discussion in case anyone can add to it.
 
The basis of my problem is this. I am trying to do co-immumo-staining by pre-incubating different fluorescent protein A conjugates with various primary rabbit polyclonal IgGs, but I always get cross-talk between the dyes, despite blocking spare protein-A binding sites with non-specific rabbit IgG.
 
Thanks,
 
Terry Morris




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