C4 or C8 instead of C18 Reverse Phase HPLC in protein purification?

Mark Dalton mdalton at worldnet.att.net
Fri Apr 18 22:15:24 EST 1997


Hi Lee
I believe that these two peaks could be separated by a dual separation 
method on the c18 column. Where by you either take advantage of the 
proteins different hydrophobicity in different pH buffers.  There was 
a method published in 1988 or 89 in JBC or similar journal by Gordon 
Gill's group where they were studying phosphopeptides from EGF 
receptors they took adavantage of this and first ran there sample in a 
phosphate buffer solution using acetonitrile elution. Then dry the 
fractions down in speedvac and resuspend in water with TFA to acidify 
the fraction or fractions with TFA.  They then reapplied this to a 
normal run using TFA water and TFA/90%acetonitrile for the elution 
buffers.  I have used this method successfully.  The greatest feature 
is when you have your peak from the second run you can simple speedvac 
to dryness to get rid of the acetonitrile and TFA. 
	One other method you could try is a cation/anion exchage column where 
the proteins may have different charges that could separate the two 
peaks.  I have no experience with this technique but it may be 
helpful.
newera at plaza.snu.ac.kr wrote:
> 
> I am trying to purify a small protein(mw 6500) using 18C reverse phase HPLC but I have failed until now because twe peaks are almost superimposed. Various grad
> Is there any chance that C4 or C8 could separate them?
> 
> 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.



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