Cyclic peptides multimerize! - How to proceed? (Jun/28/2006 )
I have recieved 18 aa long peptides that are made cyclic dy disulfid bridges between to cysteins. Now I am trying different solvents to work with these peptides. After solving them I load on tricine-gels and look at bandformation. However, what I see is ladders that seem to be bulit up by multimerisation of the peptides. The ladders can be reduced back to a single band by adding DTT so I guess that proves that it is multimerisation through disulfide bridges. What does this meen and how can I avoid getting ladders? Any suggestions are appreciated!!! The peptides are slightly positive and I have tried water, HAc, TFA, ethanol, DMSO and acetonitril. Am I missing something that is evident to some of you?
HELP!!! Please!!!
it is not so evident... can you really distinguish the different multimeric forms on the gel?
the peptide agglomeration may be due to stacking of the circles formed
you should add some methyl or acidic groups on one or two residues to avoid this pi-pi inter-molecular interactions
Thank you very much for your reply. OK, am I under standing you correctly - you meen that the laddering I see might not be due to intermolecular disulfidebridges but rather due to stacking (pi-pi) interactions. Is there some way to avoyd or minimice this happening now when I already have the peptides? Like adding something which reduces aggregation? Or is the only thing to make new peptides with methyl group om side?
I am not at all used to work with peptides and am really struggeling here.
Kind regads!
so, i'm not sure that using methanol will solve the problem...
It sounds to me your peptide formed oligomers by oxidation. You can increase the chance for intramolecular s-s formation by preparing your reduced peptide in DILUTED solution and allow it to mature. However there is no way to eliminate multimer formation.
You can design to allow intramolecular s-s formation on resin in more controllable manner; and experts in peptide sysnthesis should be able to help.
You can design to allow intramolecular s-s formation on resin in more controllable manner; and experts in peptide sysnthesis should be able to help.
Yes, but this is excactly what I have done - experts produced the peptides for me (Schafer-N Danish company) and they made it cyclic already on resin. However, when I solobulice the peptide it seem to form multimers anyway, nomatter what solution I am using. I have given some peptide for HPLC and mass and hope they will be able to see if there is monomer or multimers in it. I kind of hope that the multimerisation is something that happens in the loadingbuffer/gel and not what is acctually happening already in water.
I am out of ideas on how tho proceed.
If they measure the free SH content before deprotection/realse your peptide you would know the extent of S-S formation on resin. Maybe they did not waited long enough to allow s-s formation to complete, or maybe its your peptide design problem. I suggest that you talk to them first.