Help! Blocked N-terminus..
Shaun D. Black
SHAUN at JASON.UTHCT.EDU
Mon Jan 23 14:58:55 EST 1995
Deblocking peptides/proteins is generally a big headache. The first thing I
would do is to be sure you were not actually sequencing. That is, if you had
hit a coupld of unmodified Cys residues, these show up as blanks. Also, for
inefficient sequencers (not to say yours is this, but just in case), Ser and
Thr readily dehydrate as the ATZ- and PTH-derivatives; these, too, can look
like blanks. In other words, if your protein started with a Cys-Ser-Cys-Ser-
sequence, you might interpret it as blocked, when it was not. The cure is
to reduce and alkylate, or (my choice, initially), run at least 20 cycles.
If it is blocked, it could be acetylated, pyroglutamylated, peptide bond
rearrangement, etc. Takara Biochemicals sells a deacylase that they claim
works on proteins; it seems worth a try. Many other vendors sell pyroglutamate
amino peptidases; buy a few and try them.
As a final thought, you might consider mass spectral sequencing. On a protein
of only 10 kDa, it would be fairly straightforward (and would include the
blocking group). Have fun, Shaun
= Shaun D. Black, PhD | Internet address: shaun at jason.uthct.edu =
= Dept. of Biochemistry | University of Texas Health Center, at Tyler =
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