The protein products after expression and purification, especially during the analysis of protein products, need to be verified at the ends of the proteins to ensure that the N-terminal and C-terminal sequences are accurate. The Edman degradation method is one of the most mature methods for N-terminal sequence analysis of proteins and is widely used in the analysis of protein products.
Creative Proteomics can provide N-terminal sequencing services for purified protein products, antibodies and protein vaccines for a wide range of researchers. Using our sequencing systems, sequence information of the N-terminal 30 amino acids can be determined. Using specific protein loading systems, 60-70 amino acids can be determined at the N-terminal end.
Its basic principle is that it includes three main chemical steps: phenyl isothiocyanate (PITC) is conjugated to the N-terminal residues of proteins, phenylamino thiocyanate Formylphthalein (PTC-peptide) is cleaved, and thiazolinone aniline (ATZ) is converted to phenylisothiourea amino acid (PTH-amino acid). In each cycle, an amino acid residue is cleaved at the N-terminus of the protein, while exposing new free amino acids for the next Edman degradation, and then the protein sequence is determined by the transferred PTH-amino acid identification.
Protein n-terminal Edman sequencing services:
1D SDS gel electrophoresis of proteins (Optional)
1D SDS PAGE separation of heavy and light chains (antibodies)
Transfer gelto PVDF membranesand staining
Edman sequencing from the n-end
Determination of up to 30 amino acids (up to 20 amino acids per chain of antibodies)
Note: protein samples with a purity of more than 95% can be directly detected by spotting the protein sample onto PVDF membrane.
Sample type: dry powder or solution.
Sample concentration and purity: total amount > 200ug, purity > 90%, concentration > 0.5mg/ml.
Salt content: volatile inorganic salts < 20 mM, non-volatile inorganic salts < 5mM. Tris buffer system is not recommended, it is recommended to provide the theoretical sequence of the sample.
Although the Edman method has been widely used, there are still shortcomings. For example, the Edman degradation method cannot solve the N-terminal blocking sequencing problem (circularization, blocking), and cannot obtain the signal of the modified protein. On the basis of this technology, we added mass spectrometry, which is not limited by N-terminal blocking, and can identify post-translational modification sites of side chain groups, which is complementary to the traditional Edman degradation method.