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Immunoprecipitation of culture medium - (Jul/16/2007 )

Hi all,

I culture my cells in 5 to 7 mL of DMEM. My proteins of interest are secreted or leaked out and as such I need to do an IP with the culture medium.

Question is - Is there a problem to do IP with say 7 mL of medium since usually CL is about 500 uL to 1 mL. Also, my protein is of very very low concentration.

I read somewhere in the forum through searching the archives that I have to add alot of antibody to the medium? I was just wondering if the antibody to be added is dependent on the amount of protein I have or dependent on the amount of culture medium (or CL for that matter) I have?

Alternatively, I was thinking to concentrate medium prior to IP by lyophilisation. This will keep all the salts (and what not) from DMEM and question is - Does this affect my IP and subsequent WB?

Thanks for the help and I am very new to the field

-jaronliu-

Instead of lyophilization, you could use ultrafiltration and/or dialysis to concentrate and de-salt, then resuspend the concentrated sample in a standard IP buffer (e.g. RIPA or Tris-Tween).

-JAH-

Would ammonium sulphate precipitation help here? I'm not really sure of the overall complexity of culture medium.

-swanny-

QUOTE (swanny @ Jul 16 2007, 04:50 PM)
Would ammonium sulphate precipitation help here? I'm not really sure of the overall complexity of culture medium.



Hi thanks for the reply... I was just wondering if anyone tried freeze drying the culture medium for concentrating secreted proteins...

-jaronliu-

QUOTE (jaronliu @ Jul 18 2007, 01:04 AM)
QUOTE (swanny @ Jul 16 2007, 04:50 PM)
Would ammonium sulphate precipitation help here? I'm not really sure of the overall complexity of culture medium.



Hi thanks for the reply... I was just wondering if anyone tried freeze drying the culture medium for concentrating secreted proteins...


Hi I know there are concentrators, I used them to concentrate my proteins. The salt will go out while protein don't, it can concentrate from 30ml to hundred ul.

-rabbit-