HELP!!!! how to purify protein require for NAD

[Colin Rasmussen]

ssultang wrote:

> Hi, Everyone!
>
> I am purifying some aldehyde dehydrogenases requiring NAD with
> 5'AMP-sepharose 4B.
> But,I could not find the activity after 5'AMP-sepharose 4B affinity
> chromatography
>
> In column work,
>     - starting and washing buffer : 10mM sodium phosphate buffer
>     - eluting bnuffer : containing 0.5, 3, 7,10mM NAD
>     - and at last eluting with 100mM sodium phosphate buffer.
> Where is the enzyme?
> How can I detach the guys from 5'AMP-sepharose 4B?
>
> Please send me your idea.
> With best regards.
> Thank you in advance for your reply......

Have you checked the flow through to make sure it even bound to the
comlumn?

Colin