harsh
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synthesizing peptides
Hi guys,
I am trying to synthesize a peptide for some time now, but the yield is inexplicably low, and sometimes nothing at all. Could you answer the following
questions for me?
1.What other chemicals I can use as an alternative to NMM?
2.I use NMP as a solvent for all the reagents and for the reaction. how different it is from NMP?
Thanks!
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chemoleo
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Couple of questions- are you using Fmoc or Boc synthesis? Solid phase?
What is the peptide sequence? Did you run predictors through it?
From my experience, yields can change with solvents, but never change it from nothing to 100% or vice versa.
You can use bog-standard DMF of course, or DCM if you wish.
But really, I think your problem resides in the sequence (did you verify that this very pepitde is the only one that makes problems? How about other
peptides, which you've known to work?)
Never Stop to Begin, and Never Begin to Stop...
Tolerance is good. But not with the intolerant! (Wilhelm Busch)
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harsh
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My sequence is a fairly simple one, predominantly hydrophilic. I am using Fmoc chemistry in solid phase, using tentagel. I know this peptide doesn't
normally give problem, so it's either my bad experimental hand (which shouldn't affect so much, since most of the process is automated) or a bad
choice of reagents.
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bhassi
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well Harsh based on my experience in solid phase peptide synthesis I can suggest following points to increase the yield (these are generalized points
as you haven't given the exact sequence of your desired peptide; and most of the time it is the sequence of the peptide which makes solid phase
synthesis a nightmare):
1. always allow the resin to swallow before begining the synthesis (i.e. prior to the 1st attachment).
2. take 5 equivalents of Fmoc-amino acid for each coupling.
3. ensure the complete neutralization of the resin after piperidine mediated Fmoc cleavage.
5. never leave the peptide in dipeptide stage as it is known to cause problems due to unfamiliar reasons.
6. NMP is better than DMF for all the steps in solid phase peptide synthesis.
7. if your sequence contains problematic residue like asparagine, increase the number of couplings and also the coupling time
hope this works.
all the best
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tomcat337
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Peptide synthesis
Hi for all
I would like to learn about the cleavege and precipitation procedures. What is the best way to precipitate a peptide?
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harsh
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Actually I was about to ask the same question. Somehow, I have a feeling my cleavage protocol has some problems, because the yield is fluctuating a
lot, but mostly it is low. I would sure like some suggestions.
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nathan1
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precipitation
friends,
Use t-butyl ether to precipitate the peptide. But the selection of solvent depends on the nature of the peptide. However The hydrophobic/hydrophilic
nature, residual charge etc should be considered before selecting a solvent for precipitation
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Maya
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Almost 99.999% of Peptide Coupling Reactions are done with ( L- ) amino acids, but I was wondering if anybody had any experience with using ( D- )
amino acids instead? or any special problems associated with synthesizing these compared to the normal version. Problems with yields or special
considerations?
Reason I ask is that we are thinking of making our own peptide instead of having it outsourced to an expensive manufacturer.
It is for use in inhibitory studies since it is not degraded by biologic systems
[Edited on 16-11-2006 by Maya]
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