when we have a purified protein or antibodies and we lyophilize it to store it for long time. i just want to know that what point we should consider before performing the lyophilization procedure.
hope to get some good answers:)
I have lost very important samples with lyophilization.
Make SURE! that your sample is frozen before you lyophilize (I lyophilized a small sample and guess what? it thawed and I Lost EVERYTHING!)
Make also sure that you are under vacuum and also that you are removing liquid, not your precious proteins!
good comments, yes it is important to cool the samples first and most probably it is done with liquid nitrogen, this is very important step.
but i think there is need of some more comments so that we can optimize this process for different proteins, and also
can we use the same lyophilization procedure for different cultures for example bacterail culture or we have to follow some other way.........
Lyophilization (derived from the Greek "made solvent-loving") is also called freeze-drying, and is a method of drying that significantly reduces such damage.
Biological materials should be dried to stabilize them for storage and distribution.
Drying of biological material always causes some loss of activity or other biological damage.
Substances that are not damaged by freezing can usually be lyophilized so that refrigerated storage is unnecessary. (Important exceptions are mammalian cells, nearly all of which are destroyed by lyophilized.) Many microorganisms and proteins survive lyophilization well, and it is a favored method of drying vaccines, pharmaceuticals, blood fractions, and diagnostics. Some specialist food products are also lyophilized. They rehydrate easily and quickly because of the porous structure left after the ice has sublimed.
General Protocol for Lyophilization
Aqueous samples should be completely frozen because they may damage the pump gasket which will lead to pump failure.
ONLY use aqueous solutions in the lyophilizer (Do NOT use organic solvents)
Freeze all aqueous solutions in a -80oC freezer or in liquid nitrogen until completely frozen. It is important that solutions are completely frozen before you lyophilize.
If the sample is in container with a screw top, do not tightly screw the cap. A strong vacuum will be applied to the sample, which will crush plastic tubes if pressure is not allowed to equalize.
If the sample is in an open container, cover the top with tin foil so that the lyophilized powder is not lost when the lyophilizer tube is removed from the gasket.
Punch several small holes into tin foil allows for more efficient pressure equalization.
Edit: My comments were addressed, nothing to see here...
Thanks Kmunson. I guess I was tired.
Do not use Organic Solvents.
Make sure any aqueous liquid you are using is FROZEN as if it is not frozen, the liquid may damage the pump gasket/lyophilizer machine
I don't advise you to lyophylize antibodies! Mabs is very sensitive proteins ( like a woman) for this harsh procedure. A great loss of activity is observed in many cases. Alternative methods for Mab storage are:
solubilize Mab at high concentration (5-10 mg\ml important ) in 10mM NaPO4 150mM NaCl 10 mM borate pH 8.0 + 20% glycerine important , make aliqotes ( keep in mind that one repeated freezing - loss 15-20% Mab activity) and keep frozen at -20C.
Another exotic method is:
Keep Mabs frozen under argon at -80C.
If you've purified antibodies and decide to use protein during one - two weeks, you can add L-arginine ( up to 0.5M) in storage solution for protein stabilization in aqueous solution and 0.02% NaN3. Of course, for cell culture experiments you must dialyze protein to remove NaN3 and probably arginine , otherwise cells can't understand you :)
Good comments, it's true the sample most be frozen. You can freeze your sample with liquid nitrogen or freeze it O.N -70°C (i have done both).
Have you ever heard of lyophilization method called XEROVAC ?
I,m trying to adaptate and establish this method to preserve virtal antigen, antibodies, and live viruses. We,ve done several Trial and Error experiments.
I,m open to any information since there are only 2 journals ( worrall, and Litamoi ) that explain this method.
Thank you very much :notworthy:
cherry, im hir..alive na alive
|All times are GMT. The time now is 01:40 AM.|
Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved