C4 or C8 instead of C18 Reverse Phase HPLC in protein purification?
mdalton at worldnet.att.net
Fri Apr 18 22:15:24 EST 1997
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
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?
> 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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