chloramphenicol resin

[Paul N Hengen]

Uttam Rajbhandary (bhandary at wccf.mit.edu) wrote:

: I am trying to use a chloramphenicol resin from Sigma to retrieve CAT reporter 
: protein made in mammalian cells during transfection.  I'm having a problem 
: with elution; can't get it back. I'm using a 1M NaCl/5mM CAM elution; 0.3M 
: NaCl as recommended by Sigma will not work for TN9 derived CAT (Type I, 
: original reference notes this; Type I needs high salt to recover).
: Has anyone successfully used this resin to 
: do this from mammalian cells?

Have you considered using a nickel column to recover CAT from your extracts?
You should have a look at this article...

@article{Oswald.Rinas1996,
author = "T. Oswald
     and U. Rinas",
title = "Chloramphenicol resistance interferes with purification of
histidine-tagged fusion proteins from recombinant {{\em  Escherichia coli}}",
journal = "Anal. Biochem.",
volume = "236",
pages = "357-358",
year = "1996"}

Here's an interesting side note..I recently saw and advertisement for a IMAC
(immobilized metal ion affinity chromatography) kit in BioTechniques where the
company selling it claimed high yields when recovering a His-tagged CAT protein.
I wonder if they knew that CAT binds to nickel columns without the His-Tag???
There's a HUGE blob of recovered protein on the gel in their figure.
Slick trick guys ;-)

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