2 replies to this topic

[eva_wall]

Hello everybody,

here it is my first question. I am using Ni-NTA agarose beads to purify a protein.
According to the manufacturer the limit pressure is around 2 psi (nothing), and I am running the column to 50 psi.

Can anyone please tell me about why does the pressure limit is important when dealing with affinity chomatography?

How can this (pressure limit too high) affect my purification?

Am I destroying the beads?

I am running the column (home-prepacked tricorn) on an akta-prime.

I would really appreciate any information since I couldn't find any info on the web.

Thank you very much,

Eva

[pesji]

eva_wall, on Aug 3 2009, 12:51 PM, said:

Hello everybody,

here it is my first question. I am using Ni-NTA agarose beads to purify a protein.
According to the manufacturer the limit pressure is around 2 psi (nothing), and I am running the column to 50 psi.

Can anyone please tell me about why does the pressure limit is important when dealing with affinity chomatography?

How can this (pressure limit too high) affect my purification?

Am I destroying the beads?

I am running the column (home-prepacked tricorn) on an akta-prime.

I would really appreciate any information since I couldn't find any info on the web.

Thank you very much,

Eva

Well quite easy to understand if you press to much you will loose the ability of binding because the flow is too fast to let the Histidines residues to interact with the Ni ions I guess. Of course you might also alter physically the beady by reducing the space between them at high pressures and then change the properties. I usually run those columns at 3ml/mn and it's fine enough to purify 40ml of protein lysate in half a day

[mdfenko]

the high pressure will compress and collapse (smash) the beads, inhibit the column flow and cause further increase in pressure (causing further damage to the beads).