Affinity Purification

Joseph Polli polli~j at glaxo.com
Fri Jan 6 09:02:47 EST 1995


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Subject: RE: affinity purification

Danny,

    I have used the following protocol:

1. Prepare a preparative blot with 15-30 ug of protein on a small
minigel.
2. Transfer gel to PVDF (used for its strength and binding
capacity)
3. Stain blot with 0.2% Ponceau S for 1-2 min.  Wash blot with
water until antigen is seen.
4. Cut band from blot and cut strip into smaller squares (small
squares make it easy for elution in the small volumes used).
5. Block blot with 3% dry milk in TBS or wash with TBS (this will
also remove the remaining Ponceu S).
6. Incubate strips with serum (3-10 ml) by rocking overnight at 4
degrees C.
7. Remove strips and wash in a separate tube (usually a 6 ml
Falcon tube works well) with 50 mM Tris pH 8.0, 300 mM NaCl.
Wash 3 times for 10 min. each.
8. Elute antibody with by incubating strips with 0.5 ml of 0.1 M
glycine pH 3.0 for 5 min.
9. Remove eluate and neutralize with 50 ul of 2 M Tris pH 9.3.
Repeat step 8 with a second 0.5 ml aliquot of 0.1 M glycine.  Add
the second eluate to the first.
10. Antibody can be used directly or dialyzed against TBS/40%
glycerol to concentrate it.
11. Strips can be washed in TBS for reuse.  Store strips in 1X
TBS/0.02% sodium azide.

Good luck.
Joe
Dept. Molecular Biology
Glaxo Research Institue
Research Triangle Park, NC  27709



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