We want to order a 20-mer synthetic peptide. We are considering adding a His-tag to the 20-mer (making it a 26-mer) and ordering the crude product, and then purifying it ourselves using his-tag affinity column.
Have any of you tried this and had any positive or negative experiences, or knows of any reason why this is would be a bad idea?
Do they tell you how pure the synthetic peptide will be?
I think the problem with adding a His-tag might be the fact that any shorter/longer product impurities from synthesis will also have the same his-tag, and thus you will not be able to separate the impurities by affinity column.
I do not have any specific experience with this though. I am interested hearing more opinions.
Do they tell you how pure the synthetic peptide will be?
I think the problem with adding a His-tag might be the fact that any shorter/longer product impurities from synthesis will also have the same his-tag, and thus you will not be able to separate the impurities by affinity column.
I do not have any specific experience with this though. I am interested hearing more opinions.
Why not using FLAG-tag instead? It's 8AA in length, but has WAY higher applicability than 6xHis. Also, 6xHis can be irresponsive if dealing with highly hydrophobic peptides (didn't work in case of my Cardiolipin Synthase with just one TM domain).
As Michael suggests, this will only remove peptides which have prematurely terminated synthesis. It will not help with the more common situation where there is an internal deletion in the sequence because one step of the synthesis failed. A reversed phase HPLC is definitely the first thing to try.
Thank you all for your feedback! We will likely order the purer form. I had not considered the impurities due to incomplete peptides which still had the His-tag on it, as I did not know how abundant they were.
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