Which protein seq. lab service??

John E. Fox altabios at bham.ac.uk
Wed Jun 8 07:21:30 EST 1994


In article <Cr1Dq6.6zK at ncifcrf.gov>, pnh at fcs260c2.ncifcrf.gov (Paul N Hengen) says:
>
>Dear John:
>
>I'm thinking of detecting proteins using a dye such as Nile Red or SYBR Red
>from Molecular Probes and then slicing the protein band directly from the gel.
>Is it possible to sequence a protein directly from a polyacryamide gel without
>blotting onto PVDF? Do you foresee any problems with the red dye blocking the
>microsequencing? What is the minimum amount of protein needed for sequencing?
>
>*******************************************************************************
>* Paul N. Hengen, Ph.D.                           /--------------------------/*
>* National Cancer Institute                       |Internet: pnh at ncifcrf.gov |*
>* Laboratory of Mathematical Biology              |   Phone: (301) 846-5581  |*
>* Frederick Cancer Research and Development Center|     FAX: (301) 846-5598  |*
>* Frederick, Maryland 21702-1201 USA              /--------------------------/*
>*******************************************************************************


Dear Paul,
	I haven't used the particular two dyes you had in mind, but all the other standard
dyes such as Coomassie, Amido black, Ponceau,  etc. all work OK and don't seem to interfere.
You can avoid blotting, by cutting out the band, and extracting the protein using a 'Microcon'
filter as supplied by Amicon. (Amicon Inc    tel USA  508-777-3622     Beverly     MA.)
I haven't used one of these yet but I have just had some free samples to try out.
Amicon supply the protocol, their publication number is 311.

Our record for sequencing is 800 femto mole, 10 amino acids, but this is right at the edge
for our machine, ABI473A. 10 pico mole is fairly easy.



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