Hello, Larry,
For some reason this followup did not go back to bionet.cellbiol.
Oh well, if nobody is objecting to this thread in bionet.immunology, where
you responded to it, I guess we'll keep it there for a while. You've given
me some interesting ideas, but it's clear that I have to clarify my earlier
statements.
In article <Pine.GSO.3.96.980216145737.17907A-100000 at catbert.ucdavis.edu>,
L.A. Pike-Nobile <ez063669 at mailbox.ucdavis.edu> wrote:
>John,
>>Perhaps I'm missing something here, but if you heat-inactivate your serum
>(56oC, 1 hour) doesn't that cook the remaining B2m and make it unavailable
>for complexing with MHC I? Particularly since you have an antibody which
>recognizes an epitope spanning the two molecules, I'd think heating (and
>pehaps addition of a little 2ME) would eliminate your problem by
>eliminating the ability of B2m to complex with the a3 domain of MHC I.
>>Or am I missing something fundamental about small globular proteins?
This is an interesting idea -- maybe I *could* use ordinary fetal
calf serum if I reduce the disulfide bonds when I heat-inactivate it. It
is my understanding that beta-2m, if not reduced, doesn't irreversibly
unfold at 56 degrees. It's just a single immunoglobulin domain. But
would I also destroy some critical growth factors by doing this? Isn't
commercial fetal calf serum heat-inactivated? Do they reduce it while per-
forming the heat inactivation? As I said, a good protocol would be help-
ful.
That being said, let me remind you that I'm experimenting with
serum from the monkeys that I know are negative for antibody reactivity.
So I don't have to denature the beta-2m. If a small amount of MAb-nega-
tive beta-2m binds to my cells and reduces specific fluoresence by, say,
10%, this is far less of a problem than if some MAb-positive beta-2m
comes in, increasing my negative signal to 10-20 times background levels.
--
Rainforest laid low.
"Wake up and smell the ozone,"
Says man with chainsaw. - John Ladasky
