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.
UK.
lgbell at liv.ac.uk
