My protein getting trapped during FPLC

Nikolai Scherbak nikolai.scherbak at nat.oru.se
Tue Nov 30 16:04:35 EST 2004


Hi,

I am trying to purify a protein using  ion exchange chromatography column
Resource Q  connected to Akta Explorer 10. Protein has His-tag and FPLC is a
second step of purification after IMAC.
It is looks like my protein is getting trapped in IEC column (or just
before) and  elutes in a relatively small amount after approx. 0,4 M NaCl.
Nothing comes out during sample loading on column and nothing elutes after
wash of the column by of 1M of NaCl, 5 CV .
After cleaning with 1M NaOH I am getting a top that is up to 20 times higher
then what I am getting after elution with the buffer with NaCl.
PI of my protein is 5.3 and I am using 20mM  Tris-HCl buffer, pH 8.0.

I would appreciate very much any help or explanation for this situation.

Sincerely, Nikolai

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