Protein Profile:

Recombinant protein subunit expressed in bacteria.

Theoretical pI: 8.0.

adaptor protein.

IEX Profile:

Anion exchanger (Hitrap Q FF Column 1ml)

Running conditions:

Start buffer: 20mM tris+ 75mM NaCl or 25mM NaCl (Both were used) pH 9.0

Elution buffer: Start buf+1M NaCl. pH 9.0

flow conditions: start buffer 5ml then gradient elution 40ml 0-100%B, collected fractions: 1ml.

Also tried Cation exchanger( HiTrap SP column, 1ml)

Running conditions:

Start buffer: 50mM NaOAc+ 25mM NaCl pH 5.5

Elution buffer: Start buf+1M NaCl. pH 5.5

flow conditions: start buffer 5ml then gradient elution 40ml 0-100%B, collected fractions: 1ml.

Chromatograph:

But i found poor resolution. with broader peak.

Post-SDS analysis shown mix band.

i attached the picture of SDS and Chromatograph of Anion Exchanger.

NOTE: i tried series of variable pH buffer from pH 8-11. results are same. (ref: principle and method: Ions exchange chromatography)

i am looking for suggestions. because i struggled since whole month but couldn't get solution.

Note: i also tried to polish protein with SEC (HiLoad superdex 16/600 pg75 column). the issues with this method is 1) lost the protein (~50%) 2) poor resolution. Buffer: 50mM Tris, 300mM NaCl.