achimr at home.com
Sat Mar 27 00:41:20 EST 1999
greg moshi wrote in message <36FAF57B.7F8 at rna.bio.mq.edu.au>...
>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.
Do you really have to blot those proteins before the digest? If possible,
leave the proteins for the digest in the liquid phase.
Never dry CNBr peptides completely, always leave them in a minute amount of
liquid, depending on the tube, 20 uL can be sufficient. CNBr peptides are
often awfully hydrophobic; it is quite common that dried peptides can not be
If your chemicals are all of high quality, you can go directly into the
sequencer, without blotting the peptides onto PVDF membrane.
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.
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