In article <3A756BE7.802D7CDA at iats.csic.es>,
oswaldo <oswaldo at iats.csic.es> wrote:
> Hi there,
>> We need to use a species-specific anti-Ig Mab, mainly for
> immunohistochemistry but maybe also for immunoblotting. They are
> supplying us mouse ascites. As long as I don't have experience with
> Mabs, I was wondering what kind of purification steps are needed or
> convenient, including if we want to label it with biotin. I have read
> that ammonium sulphate precipitation maybe enough (is it an easy
Prepare a saturated solution of ammonium sulfate (use the good "enzyme-
grade" stuff). I think it's about 4 or 5 M, keep adding until crystals
persist in sitting at the bottom.
(N.B.: That's ammonium sulfate, *not* ammonium persulfate! Yes, I know of
someone who made that mistake, and no, it wasn't me)
Measure the volume of ascites you have and put it in a beaker with slow
stirring in the cold room. Slowly add an equal volume of saturated
ammonium sulfate while stirring. Let it stir gently in the cold for about
30 minutes or so more.
Spin out the ppt. at high speed (e.g., in the Sorvall at 15,000 rpm for
30 min.) in the cold.
Drain off the supernantant. Resuspend the pellet in your appropriate
buffer (PBS, coupling buffer, whatever you are using for your next step).
It may be hard to resuspend; I used to Dounce homogenize it to get it
back into solution.
Dialyze your Ab against the same buffer: at least 10 volumes, at least
Some people spin again to remove anything that didn't go into solution
(or pptd out); save the supe this time.
This should get rid of albumin (your major contaminant) as well as
various other small molecules.
nicholas_theodorakis at urmc.rochester.edu
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