Ab purification from supernatant - dialysis of large volumes (2L) (Nov/08/2005 )
I produce my antibodies from hybridoms.
I had a poor recovery and I suppose that is due to the fact that I don't equilibrate the Ab solution to bind efficiently the protein A immobilized on the column.
I would like to exchange my medium against binding buffer.
I think that I have 2 options :
1- I concentrate my cell supernatant to have a small volume and then I perform the dialisys.
in this case my problem is how I can concentrate 2L of medium in a simple way?
2- I dialyse directly my 2 liters of supernatant against binding buffer.
how can I easly dialyse 2L?
thank you.
hi,
i don't think you need to dialyse before passing your hydridoma supernatant through your column, but first check for an appropriate pH and equilibrate well the protein A
i generally pass more than once since binding capacity is limited for a given column...
if you want to concentrate your supernatant, use VivaSpin Concentrator
http://www.vivascience.com/en/ultrafiltrat...vaspin_20.shtml
avoid concentration by precipitation...
if you dialyse 2 liters make 200 * 10 ml 
Seb_
I had a poor recovery and I suppose that is due to the fact that I don't equilibrate the Ab solution to bind efficiently the protein A immobilized on the column.
I would like to exchange my medium against binding buffer.
I think that I have 2 options :
1- I concentrate my cell supernatant to have a small volume and then I perform the dialisys.
in this case my problem is how I can concentrate 2L of medium in a simple way?
2- I dialyse directly my 2 liters of supernatant against binding buffer.
how can I easly dialyse 2L?
thank you.
I agree with Tryptofan I don't think you need to concentrate or dialyse your supernatant to absorb on Protein G column except if you deal with low binding IgG subclasses check the protein G datasheet.
For the pH it's nice to check before adsorption to the column. If you still want to concentrate don't go too far except if you use a serum free medium otherwise you will end up with a very viscous solution (10%FBS concentrated) and you will reduce the capacity of your column to bind the Ab's
Pesji
Hi Seb,
Thank you for your help.
you recommand me to use the vivaspin concentrator. I used the millipore amicon ultra and I noticed that I loose a non negligible part of my protein 1rt by adsportion and 2nd when I used it more than once, the filter seems to be less efficient and let my protein going through.
how many times do you think that I can use the same unit (because I can't afford 200 units)
Just by curiosity, could you let me know why I shouldn't precipitate my antibody ? (with ammonium sulfate for exemple). because it's a widely used method.
did you try it?
Maxime.