Service Number：ABVH011
Edman degradation sequencing relies on comparing HPLC retention times of the PTH amino acids with PTH amino acid standards for amino acid assignments. The elution characteristics of the twenty common amino acids have been well-characterized, which aids in making confident assignments. Modified amino acids may present more of a challenge due to absence of standards and lack of familiarity by the protein sequencer analyst.
Custom Package
The basic analysis of Edman degradation covers the first five amino acids, but up to approximately 40 amino acids can be sequenced. The number of steps (= amino acids) needs to be determined by the client before analysis. The feasibility of longer sequencing runs depends on sample amount, sample quality and the protein's sequence. Abvigen experts can give you guidance on this topic.
1 Sample treatment
2 Loading coupling
3 Sample cutting
4 Extraction
5 Conversion
6 Identification
7 Delivery
Sample requirements
Sample amount | Minimum 25 pmol for five steps, 200 pmol for 15 steps. More sample is highly advisable. |
Sample type | lyophilized protein/peptide samples liquid protein/peptide sample in 10-100μl volatile solvent like Milli-Q water, isopropanol, acetonitrile. Check whether your sample requires cooled shipping. Protein samples blotted on PVDF membrane (PVDF membrane size: max. 3x6 mm, smaller and more concentrated sample are preferred. The protein band can be stained by Ponceau S, Amido black, Sulforhodamine or Coomassie Brilliant Blue. |
Sample purity | The sample should be as pure as possible (at least 75-80% purity) and contain only the protein or peptide. Free amino acid, primary amines, SDS, salt, buffer or other contaminants should be as low as possible since the Edman chemistry can be negatively affected. |
Cysteine modification | Cysteine without special modification can not be detected by N-terminal sequencing. Therefore the sample has to be modified for detection of cysteine. |
N-terminal blockage | Proteins and peptides which are N-terminally blocked do not have a free N-terminal amino group. Therefore these proteins can not be sequenced. More than 50% of all eukaryont proteins are blocked. On request we can perform deblocking procedures but we need a significant higher amount of protein and the deblocking does not work always, because mostly the type of blockage is unknown. |
Glycosylation and other modifications | Edman sequencing steps without the detection of an PTH amino acid, reduced peak intensity or altered retention times can be caused by glycosylation, phosphorylation or other modifications. Modified amino acids often cannot be sequenced. We recommend mass spectrometry instead. |
Short lead time
Cost-effective
The identification result of chromatography is more accurate
The reaction yield and recovery of phenyl isothiocyanate with all amino acid residues are quite high, and the reaction by-products are few
Service Process
After the two parties communicate and agree, determine the experimental plan, determine the service requirements - sign the contract - advance payment - start the experiment - deliver the results