I have been trying to make this peptide for a long time. it's a 12-mer that is basic in nature according to a peptide property calculator. After making the peptide on a PTI Tribute Synthesizer, I cleaved it off the resin for 4 hours with 88/5/5/2 TFA/Water/Phenol/TIPS. When I tried adding ice cold ether to precipitate it, no precipitate showed. Can anyone give suggestions for how to proceed? I'm a bit new to SPPS and would like some advice.
Have you tried to increase the amount of TFA in the cocktail cleavage? Maybe the cleavage process is not being enough to release the peptide from the resin.
You can keep a little bit more time for cleavage and take more amount of ether. If it does not precipitate instantly, keep at 4 deg for few hours.
In addition to all the good suggestions above, I'd suggest checking what you currently have with MALDI to see if you can see anything around or above the mass that you expect to have. That might tell you if the cleavage was successful at all.
Also, I've had similar issues with using old bottles for TFA and ether for cleavage in the past. I'm not sure why, but the reagents getting too wet in time might be a reason. If your peptide synthesis facility (or where ever you make these peptides) has these reagents ask to use theirs and see if anything changes. Chances are their reagents are fresher than yours.
The last advice is crucial it could happend that synthesis fails for some reason. If you don't have a peptide it won't precipitate. However it could be still on the resin than wahsed it by 30% acetic acid in water. What you can do also to evaporate the mixture that you already have to dence oil but not solid and than add some cold ether on it. If it will not precipate it means it is either very ether soluble (hydrophobic) or yield of synthesis is very poor. best
Hi:
Depending on the resin you have you have different conditions. Still, I would recommend you to check the catalog of Novabiochem (they provide many tricks), or check the following reference: Angew. Chem. Int. Ed., 2005, 44, 1650-1654.
Good luck
Which resin are you using, and how long do you leave the cleavage on?....then do you have any protection on the side chains which won't be cleaved by the tfa treatment (alloc/allyl, fmoc/fm, disulfide bridge, Z..)?
We have had some trouble with precipitating peptides so now we remove most of the TFA to 1-2 mls under a flow of nitrogen and then ppt with cold ether 30 mL. I don't know many peptides that are soluble in ether. If you get nothing, then re-cleave your resin with the same reagents. I have found this helpful for peptides that contain a hexa-his tag at the C-terminus which I had to cleave 2-3 times to get a decent recovery.
Also after the synthesis weigh your resin, you should have a substantial increase.
cheers
Paul
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