Binding and elution of Abs from NC bound proteins

Basavaraju Shankarappa bsh at MED.PITT.EDU
Wed Aug 11 12:21:25 EST 1993


>> I would be most interested in other people's experiences with this technique,
> > especially regarding the buffer used for elution.
> I have always used a pH 2.9 buffer, 0.1M glycine/HCl, usually with 0.1M
> NaCl added.  You have to neutralise fast, but if you have pre-titrated an
> amount of weak base, is no problem!  Always worked well in our hands, and
> takes pretty much all (ie: >70%) of Ab off first wash.  Good Ab, too -
> work in westerns and ELISA.  Can also re-use blot several times for re-
> adsorption and re-elution, so doesn't mess up the adsorbed protein too
> much either.
>  | Ed Rybicki, PhD             |       "Lord, won't you buy me        |

Another alternative that is available is incubating the membranes with
6M sodium thiocyanate.  But unlike the glycine system, you have to 
dialyze or column purify to remove the sod thiocyanate.  
Raj Shankarappa
bsh at med.pitt.edu




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