IUBio

CNBr digestion

greg moshi gmoshi at HOTMAIL.COM
Thu Mar 25 21:54:28 EST 1999


Dear Netters, I need some internal amino acid sequence of a protein I'm 
working on.  To get this, I blotted the proteins onto nitrocellulose 
membranes, excised the relevant bands and then digested the blotted 
proteins with CNBr (100 mg/ml in 70% formic acid).  After a 2 hr 
incubation at room temperature, I removed the supernatants (pooled them) 
and then lyophilised them.  The pellet was resuspended with water and 
then dried down again.  After this, the pellet was resuspended in 
reducing sample buffer and then subjected to SDS-PAGE.  Subsequent 
blotting onto PVDF membrane did not reveal any bands upon Coomassie blue 
staining.  Silver staining atfer SDS-PAGE reveals only weak, faint 
bands.  I had a hunch that some of the peptides might have been too 
hydrophobic to be eluted from the nitrocellulose membranes.  Hence, 
after CNBr digestion, the membranes were incubated with 40% acetonitrile 
(in 0.1M ammonium acetate buffer pH 9).  This treatment revealed more 
strongly staining fragments on silver stained SDS-PAGE gels, but not 
enough to blot and sequence.  Can anyone provide hints or suggestions as 
to what I could do?  Thanks in advance.


Regards,
Sham Nair
School of Biological Sciences,
Macquarie University,
NSW 2109,
Australia



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