ion exchange chromatography problem - (Dec/04/2008 )
I have a question about ion exchange chromatography.
- I have a membrane protein (pI 9.5), 13 KDa
- It's fused to MBP (maltose binding protein pI 5.1, 44 KDa) to prevent inclusion body formation during high level expresscion.
- The final protein has a pI of 6.1, 57 KDa
--> The protein is solublised in 1% TX, Tris 20 mM pH 8.0 20 mM NaCl. However, it doesn't bind to the column equilibrated in the same buffer condition. I used sigma Q sepherose
Can anyone help me with this problem? Is it because of the pI difference between the membrane protein and the fusion partner?
Try to increase pH of your buffer.
Otherwise, check the inclusion body of your protein with Urea, maybe it is not soluble after the fusion.
you can try cation exchange with sp- or cm-sepharose.
your pI may be higher than you think.