protein concentration - (Sep/26/2003 )
I have been trying to concentrate protein. Our lyophilizer showed the proper temp and vaccum settings but still my sample thawed. Any idea why? What other option do I have, as I want viable protein and my sample is in litres?
Thanks in advance
If your protein is big enough I could recommend you the method used in our lab.
I use Amicon Ultrafiltration Stirred Cells for concentrating a 24kDa protein routinely.
The largest cell, to my knowledge, holds up to 400ml solution.
I'm concentrating up to 2L of protein solution successively in 5 turns, and it works fine for me.
I use filters with 3,500Da molecular weigth cutoff (MWCO), so nothing is lost. If your protein is smaller, you will have to try it yourself once. The manufacturer says it's safe for proteins larger than 2 times the theroretical MWCO of the filter mebrane, but that could be a bit close. In my case the 24kDa protein is filtered through a 10kDa MWCO membrane. The concentrated protein is native and functional in all assays.
And you can diafiltrate your protein at high speeds with much less buffer waste after concentration...
Thanks a lot. Might try it.
you can also try vacuum centrifugation... have you heard about this?
My protein requires 20 liter buffer, but I used Ammonium sulfate to precipitate my protein into 20ml. Very beneficial, but a bit nasty. Almost all kind proteins will precipitate at 58% ammonium sulfate, and you can dissolve this pellet in 10 or 20ml of your buffer where you can store your protein. I know, you can dialyse your protein aganist PEG or sucrose. Primitively, I like concentrating peptides by steam. You put your peptide into dialysis, and hold it with clamp to holder. Then, start boiling water in a beaker under this dialysis bag, water of your peptide or protein will come out and evaporate. I do this under hood. WHile water get out of the dialysis, evaporation keep the dialsis bag enough so that you will not face temperature denaturation.
I hope, you can cope with your problem
If a sample liquefies under lyophilizing conditions despite correct vacuum and temperature settings, electrolyte balance of the solution must be restored. This is done by adding water 15%v/v. Strange that it may sound -it works just fine. Possibly the addition of water reconfigures the entire solution. You will see no more thawing.