protein recovery after anion exchange chrom

[yongyuandedj]

Hi all,
recently im trying to purify ferritin from E.coli lysate. But after anion exchange (QHP column) with 0.15M NaCl gradient, the recovery is only about 10-50%. And after size exclusion chrom its even worse. I wonder what is the general recovery for anion exchange & size exclusion. Is this normal?

[mdfenko]

the yield will depend on a number of factors including flow rate, gradient, pH, temperature, stability of protein, buffer composition, etc.

[yongyuandedj]

the yield will depend on a number of factors including flow rate, gradient, pH, temperature, stability of protein, buffer composition, etc.

Hi Thanks for your help. Theoretically ferritin is quite stable. And i changed from DEAE-column to QHP due to the limited labware. Does it mean i need to build teh gradient more slowly now coz its a strong anion exchange column? Besides, does the desalting of the cell lysis improve the recovery? If all the parameters you mentioned are optimized, can the recovery be close to 100%? Thank you very much.

[mdfenko]

first, strong and weak ion exchanger does not refer to the strength of binding. it refers to the pH range in which it will retain its anionic or cationic properties (strong has a broader range).

that being said (written?), slowing the flow will allow the protein peak to be taller (more concentrated) and the peak volume may be lower.

reducing the salt concentration of the sample prior to loading should improve protein binding.

as you improve conditions your yield should improve but keep in mind that determining the yield depends on how accurately you determine the starting amount.

[ANLA]

I agree, accurately determining how much target protein you have in the coli lysate will give the accuracy of the %recovery. Such a determination may simply be impossible.
And I agree again about that you will also have to check binding efficiency, i.e. how much of the target protein that remain unbound in the cloumn flow-through.

Also, do you mean that you used only 0.15 M NaCl for the elution? Does that give a slowly eluting ferritin peak? You could try a second elution with 0.5 M or 1 M NaCl to see if some ferritin remains bound at the 0.15 M NaCl.

When elution takes place, the concentration of desorbed ferritin may be so high in the elution zone that the solubility, under the conditions used, is exceeded. Then the precipitate (which may be irreversible) may stick in the column, including inside the pores of the resin beads.

[leekaming]

Hi all,
But after anion exchange (QHP column) with 0.15M NaCl gradient, the recovery is only about 10-50%.

I think you need to first find out where the remaining protein go. In flowthrough? aggregate inside column? in insoluble fraction? degraded?

[Uofc]

Yes, the yield of my protein did reduce more than 50% after size exclusion preceded by anion exchange. I normally do desalting of the protein which improves binding.