How to partition free amino acids from proteins in plant extract

Ricky Boernke ricky_boernke at gmx.net
Wed Aug 1 02:03:40 EST 2001



Tony Larson wrote:
> 
> Hi,
>     I am trying to quantitatively recover free amino acids away from
> total soluble proteins in a plant leaf extract.  The extract is made of
> 5-100 mg frozen leaf material homogenized in 1 mL of 80% ethanol and
> then centrifuged to remove particulates.  The amino acids in the
> extracts will ultimately be quantified by either HPLC of PITC
> derivatives, or GCMS of MTBSTFA derivatives.
>     I have heard of a method where such extracts are passed through a
> C18 SPE column to adsorb the proteins and recover the free amino
> acids in the eluate.  If this works, it would be an attractive
> alternative to using expensive and time-consuming low-molecular-weight
> cut-off centrifugation or protein precipitation methods.  However, I
> can't find a protocol for such a method using C18 SPE columns (i.e.
> rinse, load, elution conditions).  Can anyone help?
> 
> thanks
> Tony

We usually extract a leaf disc in 80% ethanol at 80 deg. C for about an
hour and use the supernatant directly for amino acid analysis. 
In case of powdered material we follow essentially the same protocol.
As you said, the particulates are removed by centrifugation and the
supernatant is directly used for HPLC. In our hands there is no need
for an extra purification step to rid of the proteins.

Ricky




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