N-Laurl Sarkosine and Hydroxy appatite chromatography

Jonathan Kurtis, MD/PhD Jonathan_Kurtis at Brown.edu
Wed Nov 21 09:12:48 EST 2001


Hi all,

I have purified a fusion protein (thioredoxin-Xa cut site-Actin binding
protein) from a Pet 32 vector using Anion exchange followed by HIC. This
protein is cut sucessfully with Xa protease, however, the ABP falls out of
solution. I have solved this by adding 0.5% N-Laurl Sarkosine to the
protease reaction. This does not inhibit the clevage and ABP remains in
solution.

I had planned to use Cation exchange to separate the ABP from the Xa
protease and thioredoxin (Thio and ABP are almost the same size- so gel
filtration is not an option). Unfortunately, Sarkosine is not soluble at
at pH 4.0 (loading pH for Cation Exhange as pI of ABP is 6.0)

I am in the process of evaluating different detergents, however I am also
considering  Hydroxy appatite chromatography.

Is  Hydroxy appatite chromatography compatible with Sarkosine (an anionic
detergent) ??? Anyone have any references ??

thanks for any help,
jake




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