protein native disulfide bonds protocols
Steven.leonard1 at ntlworld.com
Tue Aug 6 11:27:51 EST 2002
You could try a search for Schofield and glutathione I forget the paper
""Baichen Zhang"" <Zhang at mpimp-golm.mpg.de> wrote in message
news:0F072619A2C4594781FBFF69DA96AA379DE8 at MAIL.mpimp-golm.mpg.de...
Could you tell a reliable protocol on trapping, stablization and
localization of protein/protein complex native disulfide bonds? Monitoring
the dynamics of protein/protein complex will be a luxurious gift.
I hope this is not a luxurious request.
Von: tmorris at uhnres.utoronto.ca [mailto:tmorris at uhnres.utoronto.ca]
Gesendet: Donnerstag, 1. August 2002 18:18
An: proteins at hgmp.mrc.ac.uk
Betreff: How does protein A bind IgG...really?
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:
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.
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
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