Can those of you who have sequenced 2D blotted proteins (these went onto PVDF in
Towbin buffer) prepared from the Pharmacia: Immobline DryStrip and ExcelGel XL
SDS system (or those who aren't but have good thoughts anyway) offer experiences
with cysteine containing sequences?
I received a blot of a whole cell bacterial lysate and was asked to sequence
several proteins. (I'm using a 494 clc.) The data analysis presented many
challenges and in the course of requesting more detail about the sample
preparation, the researcher told me that the pre-cast Pharmacia gel protocol
includes Iodoacetamide in the sample buffer. Does anyone know if it generates
identifiable PTH CAM Cys (i.e do I have to worry about all by E calls)?
One multiple component sequence contained a large peak in amino acid cycles 1
and 2 at the DMPTU position. I don't think I've sequenced a PTH-Cys
S-propionamide since ABRF-97SEQ but would like to know if the Pharmacia system
generates this acrylamide product (and I can suggest C in those positions) or if
there is another modified amino acid that may be eluting here.
This bacteria is from the Rhodothermus family. Is anyone aware of modified amino
acids in this species?
Best regards,
Linda
Big Apple Minute: I was commiserating with a friend about the frustration of
living in NYC and never having the time to enjoy its pleasure, except that is if
out of town guests come in and we get to show them around. So come and visit me!
Really, if you can do without first class accommodations, my finished basement
(shower even) has a mattress on the floor. Although I haven't used it myself, my
daughters teen aged friends who sleep over don't complain.
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Linda Siconolfi-Baez
Core Protein Sequencing Center
SUNY Health Science Cntr.
450 Clarkson Ave.
Box 8
Brooklyn, N.Y. 11203
718-270-1267 (phone)
718-270-3316 (fax)
lsiconolfi-baez@netmail.hscbklyn.edu
http://www.hscbklyn.edu/SUNY/Biochem/Bioparts/ProtSeq.html
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