Lyophilization - concentrating after FPLC - (Jan/22/2007 )
Hi,
when you think you have finished something and you only want to do the very last step sometimes you find yourself in front of a totally new problem. It does not matter if its big or small, its new. So here it comes:
I lyophilized my protein (staphylococcal lipase) that has been eluted in around 15 ml buffer and adjacent desalted (total end volume ~ 30 ml). After lyophilzation its hard to dissolve the powdered protein again - at least perhaps 85% did not go into solution anymore.
So how does it go on? What shall I do now? Is there a strategy what factors I should check first?
thanks for answering!
what do you need to do with the protein?
if you are going to run it on sds page then you can suspend in sample buffer.
you can suspend in urea or some detergent (besides sds).
maybe the protein requires some salt to suspend (like myosin).
pH?
when you think you have finished something and you only want to do the very last step sometimes you find yourself in front of a totally new problem. It does not matter if its big or small, its new. So here it comes:
I lyophilized my protein (staphylococcal lipase) that has been eluted in around 15 ml buffer and adjacent desalted (total end volume ~ 30 ml). After lyophilzation its hard to dissolve the powdered protein again - at least perhaps 85% did not go into solution anymore.
So how does it go on? What shall I do now? Is there a strategy what factors I should check first?
thanks for answering!
Well a lot of proteins when you by them lyopholized have lots of other stuff in them...citrate salts, Peg etc. Your typical roche Glutamate dehydrogenase will only be 5% protein so I suppose that means you cant always just lyopholize what you like.
YOu could try ammonium sulpahte precipitation as a storage mechanism and then just use a pd-10 column or dialysis when you need to use some.
jc
thanks a lot for your suggestions!
another point: Could it be that due to deep freeze the protein at -80°C that the protein could precipitate itself after thawing? that the structure is somehow affected?
I'm doing this because I need antibodies against my lipase. Therefore I wanted the protein a) as clean as possible and 
in powdered form for easy calculating the amount of protein that is needed for an immunization.
As an alternative, couldn't I run a native gel, cut out the protein band and give it away for immunization anyway?
How would you concentrate a protein before you give it away for immunization?
another point: Could it be that due to deep freeze the protein at -80°C that the protein could precipitate itself after thawing? that the structure is somehow affected?
I'm doing this because I need antibodies against my lipase. Therefore I wanted the protein a) as clean as possible and
in powdered form for easy calculating the amount of protein that is needed for an immunization. As an alternative, couldn't I run a native gel, cut out the protein band and give it away for immunization anyway?
How would you concentrate a protein before you give it away for immunization?
you are right: freezing of proteins in water may affects its structure up to denaturization; so use freezing buffers; lyophilized protein is only faced with freezing damage if it is not really dry