Has anyone ever had lousy recovery of Histidine--and what are the tricks
to improve yield. I understand that the cleaved His remains up in the
reaction chamber when you use a glass fiber filter--and that changing the
argon dry (I use my trusty old ABI 470) just after the liquid pulse of
neat TFA--will bring more His down into the flask--but also, probably,
wash off more protein from the GFF.
I put some BSA onto a Prosorb and also see low yield of His.
For proteins that have been run on gels and then electroblotted to PVDF,
is it common to see low His yields? Does His get alkylated on gels?
On the column, I use Premix on A with 3.5% THF and basically the B2
molbile phase. I've kept the His peak eluting to the right of the Ala
peak, as I was having problems getting sufficient separation between the
peaks when I had it positioned to the left of Ala. The His peak is still
a fairly fat, low peak; but I've fairly good at seeing the lift off of
the baseline that is His.
Thanks for any advice,
Deb McMillen
Institute of Molecular Biology
University of Oregon
Eugene OR 97403