Kang Ke-Won (kwkang at sorak.kaist.ac.kr) wrote:
: I have a problem on sequencing a purified protein. MW of the protein is
: about 10kd and has thrombin inhibiting activity. While sequencing the protein,
: unusual amino acid peak appeared in first cycle and then no peak appeared there
: -after. I used TFA method to deblock the protein. After deblocking, same
: unknown peak was detected in first cycle and then many of peaks were detected.
: The protein was blotted to PVDF membrane and sequenced by automated sequencer.
: Is there a good method to deblock N-terminus without fragmentation?
: Please Help!
Unfortunately the methods available to un-block (usually acetylated)
proteins do give very clear data when subsequently sequenced. I would
suggest carrying out an in-situ digestion of the protein on the PVDF
membrane or alternatively an in-gel digestion of the stained gel.
Following HPLC separation of the peptides you can obtain some great data
from the sequence of the individual peptides. Both methods give excellent
results. Please feel free to E-mail me if you need further info about
these methods.Good luck.
Robin J. Philp
Protein Chemistry
Institute of Molecular and Cell Biology
National University of Singapore
E-mail: mcbrp at leonis.nus.sg
