I believe that excessive drying after the TFA cleavage step will produce
low His and Arg recovery.
Dick
>Hi, all,
>
>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