Help with concentrating proteins in culture medium - (Aug/25/2005 )

Hi

I want to concentrate PAPPA in culture medium using Microcon YM10 from millipore. My concern is will the protein retain its activity after centrifugation (concentration)? I would appreciate any help from anyone who has done this or something similar before?

Hi,

It's based on your protein's MW, solution volume, spin time etc. Usually, recovery is good, you do not have to worry about it.

good luck



Hi

I want to concentrate PAPPA in culture medium using Microcon YM10 from millipore. My concern is will the protein retain its activity after centrifugation (concentration)? I would appreciate any help from anyone who has done this or something similar before?

Thanks very much for your response. I want to follow up on the first question about concentrating PAPPA in culture medium. Since the salts and other small molecules that were in the culture medium will have left, will my protein be happy (retain enzyme activity) in the remaining volume of medium (of unknown composition, pH etc)? I have been wondering if after the initial spinnging I need to add some buffer and spin again?



[I want to concentrate PAPPA in culture medium using Microcon YM10 from millipore. My concern is will the protein retain its activity after centrifugation (concentration)? I would appreciate any help from anyone who has done this or something similar before?]

before concentrating your protein, make a dialysis against the appropriate buffer (PBS or NaCl)

i've done such experiment with a mAb and it has retained all its biological activity

Seb_

The mechanisms are different.
As you are using membrane technology rather than salting out, the structure of the protein should not be a problem most of the time.

Membrane filtration is one of the best methods to maintain the "happiness" of the protein.
Salt and buffers would be filtered out of the membrane.

What is the size of PAPPA?
Please make sure you have chosen the right cut-off.
Protein with 30kDa will choose YM-10 while 300kDa will choose YM-100.

If you have chosen higher cut-off, the filtration rate is faster, but the recovery will be lower.