low pH elution of protease from pepstatin-Sepharose

Tim Krieger tkrieger at interlog.com
Wed Sep 4 22:30:12 EST 1996


osullida at SUN1.BHAM.AC.UK ("Deirdre O'Sullivan") wrote:

>Hi,

>I am trying to figure out how to elute pepsin and other acid proteases
>from pepstatin-sepharose in active form. Methods using high pH (> about 7) irreversibly
>inactivate the enzymes. Other methods use chaotropes.

>I can elute about 50% of bound matter using a step change of NaCl from 0 to 1 M.
>That still leaves a lot of protein on the resin !

>Any assistance appreciated !
Have you tried collecting a 50mM Tris-HCl, pH 7.5, 1M NaCl eluent into
test tubes containing an equal volume of 100mM Acetate buffer, pH 4.5?
The rapid acidification upon elution sometimes improves yields
dramatically.

Tim Krieger, Ph.D.
Connnaught Laboratories/PMC






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