Cleaning up primary antibodies??

Nick Theodorakis nicholas_theodorakis at urmc.rochester.edu
Wed Dec 13 11:56:36 EST 2000


In article <Pine.A41.4.10.10012130206310.200928-
100000 at acs4.acs.ucalgary.ca>,
  Neal Robert Melvin <nrmelvin at ucalgary.ca> wrote:
> Is it possible to 'clean up' a primary antisera by incubating with protein
> A, and then somehow gently eluting the IgGs from the protein A to be able
> to use?? What is the best way to go about this??
>

You could do this; Pierce sells a Protein A "Gentle Elution Buffer,"
which is supposed to keep the IgG from denaturing.

Also, here are some older methods for partially purifying Abs from serum
or ascites:

Ammonium sulfate cut: Add ammonium sulfate to 50% saturation -- most
easily done by adding an equal volume of saturated amm. sufate. soln.,
add slowly while stirring in the cold. After about 30 min, spin out the
ppt, resuspend in PBS or whatever you like, dialyze out the residual amm.
sulfate, and Bob's your uncle. IgG ppts out, but albumin (the principle
contaminant) should remain in the supe you through away. This is most
easily done if you have alot of serum (like a rabbit bleed-out, e.g.)

Blue-Sepharose chromatography: Albumin binds very tightly to Cibaron
Blue, but IgG will pass through the column.

You could also try chromatography on DEAE, but that's something you may
not want to get into if chromatography is not your thing.

Nick

--
_______________________________________________
Nick Theodorakis
nicholas_theodorakis at urmc.rochester.edu


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