Pressure limit AND Ni-NTA beads

[Pepa Florez Pérez]

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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

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[Dr Engelbert Buxbaum]

Am 03.08.2009, 14:25 Uhr, schrieb Pepa Florez Pérez  
<snipeurope@...>:


> 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?

When you expose your beads to too high a pressure, they get compressed,  
and as a result packed more tightly. That reduces the flow rate of the  
column. You may also affect the pore size of the gel, so that fewer active  
groups are accessible to the protein -> reduced capacity.

If I did the math correctly, 2 psi corresponds to about 0.13 bar or 1.3 m  
of water column, which is low even for makeshift setups with gravity feed.  
Probably simple agarose. I would switch to a stronger (crosslinked) gel  
like Sephacryl.
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