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Set up meetingProtein characterization for optimization of manufacturing processes.
Explore customer caseOptimized HPLC analysis of peptides for clinical trials and stable GMP production
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The Edman degradtion chemistry is a cyclic procedure: It couples the PITC reagent to the free N-terminal amino group and then cleaves off the protein. Next, the PITC coupled residue is transferred to a flask and converted to a PTH-residue. Finally, the HPLC chromatography identifies the amino acid. The Edman reaction cycle is repeated until a sequence of N-terminal amino acids is determined.
Since the Edman sequencing process never is 100%, the signal intensity decreases as the process goes a long. Therefore, the amount of material needed also increases with increased number of residues.
The Edman sequencing analysis will not work if the N-terminal amino group is blocked for the PITC chemistry. E.g. due to acetylation or pyroglutamic acid, which is frequent for antibodies. If the sample contains more than one protein, the chromatography then shows multiple amino acids, and a unique sequence can thus not be read.
N-terminal HPLC chromatogram distingushing all 20 amino acids except Cysteine
Results from 5 cycles of N-terminal proteins sequencing by Edman degradation including a blank and a standard
The analyses are performed on PVDF membrane bound proteins or pure in-solution samples.
Use this guide for help on How to convert your sample to PVDF membrane-bound proteins. Please send the PVDF membrane samples as stained bands that are individually cut out from the membrane and placed separately in high-quality tubes. You can also ask us to run the 1D gel electrophoresis and transfer to the PVDF membrane.
Protein sequencing analysis by N-terminal Edman degradation generally requires 30-50 picomoles of pure protein or 200-250 picomoles of antibody (if the antibody requires conversion to PVDF membrane).
If a protein sample contains more than one protein, the analysis shows a mixture of amino acids in each Edman cycle, and characterization can become too complex to yield proper sequence data.
1D SDS PAGE and blot onto PVDF membrane before N-terminal protein sequencing
