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Re: [ccp4bb]: NAD-binding Proteins
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Try loading at a low pH followed by a pH gradient to a higher value.
On RedA (Amicon), pH 6.5 (loading) to 8.5 worked well for Nucleoside
hydrolase from C. fasciculata.
Hope this helps.
Chris Snook wrote:
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>*** CCP4 home page http://www.ccp4.ac.uk ***
>I now that this is not a CCP4 question, but I need some help/advice with
>regard to Affinity Chromatography of an NAD binding protein.
>I am using Bio-Rad's AffiBlue gel but am unable to get the protein to bind to
>the column. Extensive dialysis, with and without the substrate, fails to
>elicit binding to the column. The protein is loaded in low ionic strength
>Does anybody have a preference of gel type for for this type of column? Are
>there any tricks to aid binding to the column?
>Many thanks for any help
Deshmukh N. Gopaul, Ph.D.
Departement de Biologie Structurale et Chimie
Laboratoire de Biochimie et Biophysique Des Macromolecules
Bat. Metchnikoff, p3011 email: firstname.lastname@example.org
25 Rue du Docteur Roux Tel. (+33) (0)1 184.108.40.206
75724, Paris Cedex 15 France Fax. (+33) (0)1 220.127.116.11