Proteins recover from different buffer - How to obtain again proteins from ST buffer? (Oct/20/2008 )
We have a problem in our lab, due to the fact that a person working on this project doesn't work here any longer...
In order to obtain proteins from patients and normal controls' urines (suitable for IEF) we used to precipitate by adding 2 vols of acetone o/n at -20C and centrifuging at 12000g for 10 mins at 4C. Thus we used to resuspend the pellet with ST buffer (sucrose 250mM, Triethanolamine 10mM) and store it at -80C.
On day of IEF, we take an aliquot of pellet in ST buffer containing enough proteins and we add Rehydratation buffer (urea 7M, thiourea 2M, CHAPS 2%, DTT 50mM, ampholines 0.2%) until the volume of 300ul. Rehydratation buffer is 75% of total volume.
Due to many unsuccessful isoelectrofocusing sessions, we decided to try removing ST buffer by resuspending the same proteins in ONLY Rehydratation buffer before loading.
My question is which is the best way to recover proteins from ST buffer, so as not to loose all of this stored material. Would it be safe to re-precipitate with acetone, following the same previous protocol?
Any of you have ever used proteins in ST buffer for IEF, obtaining any success?
Thanks a lot,
Are you sure the failure of IEF has been because of the ST buffer? How has IEF been "unsuccessful"?
If you do choose to recover the samples from the ST buffer, I would test the system first with an expendable sample. You can take a normal sample, spike it with some proteins that you expect to find in the clinical samples, take it through the procedure, both with and without ST resuspension, and see what kind of results you get on IEF. This will also give you some idea of losses through the procedure, if you haven't already done that.
"Unsuccessful" means that the IEF stopped and couldn't go any higher than 5,000 V/h, so it couldn't go to the next step.
Any of you have ever had to handle these buffers?