Dissociating agents to prevent protein aggregation

antonio at liverpool.ac.uk antonio at liverpool.ac.uk
Mon Mar 3 12:57:54 EST 1997

newera at plaza.snu.ac.kr wrote:
> I am purifying a small protein(mw 6100), which tends to aggregate well during its purification process although the completely purified product does not; so I
> In choosing dissociating agents, some points should be considered :
> 1. My protein seems to be quite easily renatured, because the last step of its purification is reverse phase HPLC(water:acetonitrile) and freeze-drying. In add
> 2. I am using a cation exchange column(BioRex 70) and an affinity chromatography(Blue Sepharose); the less the agents affect the chromatographies, the better.
> 3. The buffer throughtout the process is 20mM Na phosphate, pH 6.8, no salt.
> 5% ethylene glycol is considered now.
> Is EDTA, DTT or 2-mercaptoethanol helpful?
> If you give me any advice on this matter, I will much appreciate it.
> Thanks.
> Lee, Ji Hyun
> --
> email   : newera at plaza.snu.ac.kr
> address :
>   Lee, Ji Hyun
>   Laboratory of Physical Pharmacy(Prof. Lee, Bong Jin)
>   Seoul National University
>   College of Pharmacy
>   Shinlim-Dong, Kwanak-Gu
>   Seoul 151-742, Korea.

I am aware of some peptides (MW 4330) which readily aggregate at around mid-range pH but 
can become very much more soluble and stable at others. So you could try changing the pH 
to 3 or 8, though this would require some test runs to sort out changes to your cation 
exhange purification system. 

Perhaps something as simple say addition of non-ionic detergent, such as triton-X to 
your buffer systems would overcome your problems.

DTT and mercaptoethanol are only likely to be very useful if your target protein has 
cysteines or methionines in it.

I hope this helps,

Len Bell.
Liverpool University.
lgbell at liv.ac.uk

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