I wanted to quantify the amount of protein present in three different bands.
They do not transfer equally to pvdf so I thought to run AAA on the gel
slices. I followed the description provided by Williams and Stone in TIPC
VI p144 for AAA on gel bands that used Post-Column analysis.
My instrument, however, is the Agilent 1100 (nee HP) using automated
AminoQuant chemistry of OPA and FmocCl for Pre-Column analysis. I
couldn't find any reference to the use of Pre-Column analysis of a
hydrolyzed protein sample in gel but forged ahead anyway.
I extracted the hydrolyzed plugs with 2x100 uL 0.1N HCl, dried the combined
extracts and redissolved in 25 uL 0.4N Borate buffer. One and 5 uL samples
were derivitized and then injected.
The results: very little amino acid recovered including added Norvaline and
Sarcosine. There was one large peak in the middle of the chromatogram
emerging just after Nva (but too large to be Nva). The same
hydrolysis/extraction procedure (using 30% MeOH/0.1N HCl) was done for
protein samples on pvdf and the pvdf samples furnished the expected amounts
of amino acids.
Has anyone done AAA on gel slices followed by Pre-Column derivitization?
Shouldn't the amino acids have been extracted as per Williams and Stone? (I
also extracted three samples with 30% MeOH/0.1N HCl with the same results.)
Was the AminoQuant chemistry overwhelmed by the gel hydrolysis products?
Any experiences or thoughts on this problem would be appreciated.
Dr. Jim Farmar
Laboratory of MicroChemistry
Lindsley F. Kimball Research Institute
The New York Blood Center
310 E. 67th St., Rooms 3-82 & 3-85
New York, NY 10021
212 570-3128 (v); 212 570-3195 (f)
For information about the Laboratory of MicroChemistry at NYBC or for
request forms, please visit:
http://www.nybloodcenter.org/framesets/FS-3C17.htm
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