Glycerol stored culture supernatants

redeamer alion85 at hotmail.com
Fri Jun 18 07:22:34 EST 2004


l.roddam at mailbox.uq.edu.au (Lou Roddam) wrote in message news:<BCF60649.208A%l.roddam at mailbox.uq.edu.au>...
> Hi,
> 
> I am doing ELISA analysis of monoclonal culture supernatants that have been
> stored in 50% glycerol. The glycerol is interfering with the ELISA but I do
> not want to dilute my supernatants too much. How can I get around this???
> Would incubating at a higher temperature help?
> 
> LR

Hi Lou!

You can dialyze your supernatants against the buffer of your choice.
Antibodies are very highly soluble substances --> do not afraid of
precipitation or something like that... I suggest you will have some
2X volume after the dialysis is complete (V[final] ~ 2 * V[initial] is
not big at all).

Another alternative is to do western blot (its when you put one sample
on the whole SDS PAGE gel and a marker --> then transfer to membrane
--> the cut the membrane into collateral fragments and with each
fragment you perform western blot with different antibodies) <-- this
is good for LINEAR EPITOPES. Because your hybridomae supernatants'
dilution will hardly be less than 100X glycerol wont interfere
anymore.

If you want to retrieve conformation sensitive epitopes then you have
an ISSUE and should consider dialysis. Or for example, such technique
as Immuno Precipitation --> there your dilution will be too big for
glycerol to take any effect.

Best wishes,

-------> Drew



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