The N-terminal sequence of peptides and/or proteins can be carried out using the EDMAN degradation method on samples purified by HPLC or by SDS-PAGE (either monodimensional or bidimensional), followed by electroblotting on PVDF (PolyVinylidene DiFluoride) membranes.
Pre-treatment of the sample of the cysteine residues, is also performed, (i.e. reduction and alkylation).
The sequence of the first 10-20 residues at the N-terminal of a protein gives sufficient information to allow the following:
- assigning the site of proteolytic maturation
- synthesizing oligonucleotide probes for specific cDNA isolation
- identifying the protein in databases
- aligning the protein against DNA sequences
- assessing the purity of the peptide/protein
Primm can also provide:
1. Preparation of samples for N-terminal sequencing
Separation of protein samples by SDS-PAGE and blotting onto PVDF membrane.
2. Separation of peptides for internal N-terminal sequencing
Purification of protein samples by HPLC or SDS-PAGE, enzymatic hydrolysis, fractionation of peptide mixtures, MALDIMS analysis of selected fractions.
Homogeneous peptides are submitted to automated Edman degradation
Important for sample preparation
See technical info (PDF General information – Guide Lines for N-terminal sequencing)
- avoid the presence of primary or secondary amine (es. do not use Tris buffer)
- avoid high concentration of detergents and glycerol or non volatile salts
- avoid Tris-Glycine buffer during blotting of the samples
- use ONLY PVDF membrane for blotting procedures
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