Frequently Asked Questions
How much sample do I need to get sequence information?
Assuming a fairly pure sample, 0.5 – 1.0 pmol of sample is desirable. Sometimes it is difficult to quantify this amount of protein. As a rule of thumb, if you can see a band or spot on a gel stained with colloidal coomassie, there is usually sufficient material to obtain a sequence.
What is the identity of this protein or peptide?
Usually, identification of 10 or less residues is enough to conclusively identify a protein. If MS data is also available, this can add confidence to this identification.
I have limited sample purity available. Can you still identify this protein?
Even if multiple species are present in the sample, computer algorithms are available to deal with this, and usually identify the major species present.
I have a sample that is N-terminally blocked. Can you get internal sequence information?
A gel plug or a protein in solution can be cleaved chemically or enzymatically, and the peptides can be separated and sequenced to obtain an internal sequence.
I have a synthetic peptide that was analyzed by HPLC and MS. Is this enough information to be sure of peptide identity?
These techniques do not verify that the sequence is correct. An incorrect sequence can still have the "desired" or correct molecular weight. Edman sequence analysis will confirm this without a doubt.
Can you estimate the purity and concentration of my protein?
Edman sequencing can give definitive information about sample purity, and concentration can be given with about a 5% – 10% error. Amino acid analysis is still the "gold standard" for the most accurate determination of protein or peptide concentration.
How long does a sequence analysis take?
Usually, a 20 amino acid sequence can be run overnight.
What is the maximum number of residues that may be identified?
Given enough sample, and reasonable purity, our laboratory has successfully sequenced up to about 60 amino acid residues.
What does it mean if I don't get any sequence data?
Usually, this is due to one of two possibilities: either the sample is N-terminally blocked OR there was not enough sample present. Sometimes, there will be clues present to indicate which of the two is the cause. An N-terminal blocked sample will exhibit increasing sample noise or background as the sample sequences. Not enough sample present generally has no increasing sample background as the sample sequences.
I have an unknown protein sample that I want to identify with confidence. Should I get N-terminal analysis of MS?
This is a complicated question. Please contact Mark K. Garfield directly to discuss your particular project.
I want to identify post-translational modifications of my protein sample. What is the best way to do this?
Again, this is a complicated question. Please contact us.
back to top