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Since we had conducted the NCL earlier and subsequently in the complex systems, we are unable to explain the breakdown in the case at hand. Clearly more work is necessary to define the scope and limitation of NCL in highly complex settings. Conceivably we are operating in peptide sequence with 27 and 28 which are particularly resistant to the methodology which has been developed. It also possible that the particular glycol-domain assembled in this case serves to shield the proposed site of ligation. These issues are being evaluated to allow a more definitive statement as to scope and limitation of NCL in these settings.
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