In article <3f9bn3$i07 at mserv1.dl.ac.uk>, psansom at hgmp.mrc.ac.uk (Mr. P.A. Sansom) says:
>>Iodinated peptides spread on RP-HPLC. Why?
Some of your message got lost so you may have covered some points I raise.
>Why don't I get sharp HPLC peaks with the labelled peptides? Is it
Do you get good peaks with the unlabelled peptides? If so then you can
discound equipment i.e. any connections post collumn which are not
good, i.e. a gap is created between one bit of tubing and another
can cause some mixing and therefore peak broadening.
>(1) my equipment?
See above.
>(2) because the label is incorporated into such a small proportion of the peptide? I estimate 100pg is labelled per ug starting material, and my assay sys
> Shouldn't have any effect (I think - somebody correct me if I'm wrong)
>(3) my gradient protocol? I go from 5% to 45% acetonitrile
>over 20 minutes. Shallower gradients don't improve things,
A steeper gradient is what would give sharper peaks. However your current
gradient looks good. As you're using a RP-C18 column you should get fairly
sharp peaks.
>Why do I get multiple peaks? Is it
>>(1) my peptides? I'm new to this lark so I've got no criteria to judge the quality of the peptides by other than the synthesis report and the say-so of th
The profile of unlabelled material needs to be looked at for this one.
are the many peaks present in the unlabelled sample - lots of differing
peptides, or do they appear after Iodination?
>>(2) my labelling protocol is not as good as I think it is, and I'm getting incorporation of 125-iodine into the histidine residues? I must say that the di
No idea
>Also, does incorporation of iodine affect the pI of a peptide?
No idea
Hope this is of some help.
(BTW I used to work at the Kennedy - say hi to Mike, Jay et al)
Bob.
================================================================
Bob Lauder R.Lauder at lancaster.ac.uk
Postdoctoral Research Asscociate Tel +(44)(0)1524 65201
Division of Biological Sciences Fax +(44)(0)1524 843854
Lancaster University, Lancaster, UK
